Patent Description:
Nanopore sensors have been developed for sensing a wide range of species, including single molecules such as polymer molecules. A known nanopore sensor device is a MinION™, manufactured and sold by Oxford Nanopore Technologies Ltd. The nanopore-based sensing therein employs the measurement of ionic current flow through a biological nanopore located in a highly resistive amphiphilic membrane. The MinION™ has an array of nanopore sensors. As a molecule, such as a polymer analyte e.g. DNA, is caused to translocate a nanopore, measurement of the fluctuations in ionic current may be used to determine the sequence of the DNA strand. Nanopore devices for detection of analytes other than polynucleotides such as proteins are also known from <CIT>.

An alternative to biological nanopore devices, such as MinION™, are solid state nanopore devices. <FIG> shows a portion of a single sensor device <NUM> with a solid-state nanopore <NUM> disclosed in <CIT>, in which an analyte <NUM> passes through a body <NUM> from a cis reservoir <NUM>, through the solid-state nanopore <NUM> and into a fluidic passage <NUM>; a signal is read via a sensor <NUM> located close to the solid-state nanopore <NUM>. Electrodes <NUM> are provided in the cis and trans reservoirs <NUM>, <NUM> for inducing the analyte <NUM> through the solid-state nanopore <NUM>. In one example, <CIT> discloses a device for nanopore sensing, said device comprising: a structure arranged to separate an analyte reservoir and an outlet chamber, the structure comprising: a nanopore layer incorporating a solid-state nanopore and a base layer incorporating a channel, the nanopore layer and the base layer being sandwiched together such that the nanopore and the channel are aligned to define a nanopore structure that comprises a passage defined by the nanopore of the nanopore layer at one side of the passage and the channel of the base layer, at the other side of the passage, wherein the passages provides fluid connection through the structure between the analyte reservoir and outlet chamber; drive electrodes connected respectively in the analyte reservoir and the outlet chamber for imposing an electrical potential difference across the passages; an electrical transduction element that is exposed to the passage for measuring the fluidic electrical potential at the electrical transduction element.

The performance of solid-state nanopore sensors is limited by the sensing components, manufacturing techniques and their tolerances, which can occur as a result of variation in the formation of the nanopore or the assembly of the sensor. These and other factors detriment the bandwidth, sensitivity and ability to control such nanopore sensors.

The present invention is concerned with overcoming problems associated with implementing a nanopore sensor array having a plurality of nanopore sensors.

The present inventors sought to improve upon known nanopore sensing devices by providing the ability to control the movement of an analyte while also improving the measurement accuracy by mitigating factors that impede on the measurement, such as noise caused by parasitics and contaminated sensing components. Moreover, the improved devices allow the nanopore structures, and nanopore sensors implemented therefrom, to be formed in large arrays in an efficient manner without inhibiting the control or performance of the array.

In a first aspect, the invention provides a device for nanopore sensing as defined in claim <NUM>.

When provided with a fluid, such that there is a fluid connection between the drive electrodes and the nanopore structure, then the control signal applied to that nanopore structure can alter the electrical potential difference across that nanopore structure with respect to the drive electrodes.

When provided with a fluid, fluidic electrical potential can be measured at the electrical transduction element. When provided with a fluid, the fluidic electrical distribution around that nanopore structure can be altered.

In operation, fluid resides in the analyte reservoir, outlet chamber and passages of the device wherein the reservoir and chamber are fluidically connected. The fluid in the reservoir, chamber and passages of the nanopore structure can be different fluids.

Exemplary nanopore structures that may be used to support a nanopore are disclosed in <CIT>. Examples of nanopores that may be supported by the nanopore structure are biological nanopores such as protein nanopores.

The analyte reservoir can function to receive an analyte for sensing by the nanopore array. The outlet chamber can function to receive an analyte that passes through the nanopore array.

The nanopores separate a cis side and a trans side of the device. The analyte reservoir may be considered as the cis side of the device and the analyte outlet chamber may be considered as being part of the trans side.

The device may be provided with or without fluid. The fluid in the analyte reservoir, outlet chamber and passages of the nanopore structure can be different fluids.

Each nanopore structure in the array of nanopore structures can be considered a pixel, each pixel comprising an aperture, an electrical transduction element and a control terminal. An array of pixels can be arranged as a rectilinear grid in a manner analogous to the arrangement of pixels on a television screen. A nanopore in the nanopore structure forms part of the passage, namely a section of the passage of nanometer width. The nanopore may be biological nanopore supported in an amphipathic membrane. The amphipathic membrane may be supported by pillars such as disclosed in <CIT>. When the analyte reservoir, outlet chamber and passages of the array of nanopore structures are provided with a fluid the drive electrodes can impose an electrical potential difference across the passage. The drive electrodes provide a potential difference across the apertures to induce passage of a charged analyte through a nanopore of the array. The potential difference can be altered to change the speed or direction of translocation of the analyte.

Each electrical transduction element in the array functions as a sensor electrode. Changes in ion current flow through the nanopore cause fluctuations in electrical potential caused by changes in ion current flow, said electrical potential may be measured to determine the presence or a property of an analyte. The fluid in the device, which may be aqueous, may contain ions Multiple analytes may be translocated.

The drive electrode serves to provide a common potential difference across the array of nanopores, wherein multiple analytes may be measured simultaneously in the array. Measurements are made at the electrical transduction elements in each nanopore structure.

In accordance with the invention, each nanopore structure has an associated control terminal. This control terminal is an independent connection to a control signal generated externally from the structure. This allows the electrical potential to be applied independently of altering electrical potential differences across other nanopore structures in the array.

The control signal can be generated within the nanopore structure in response to an external trigger or switch. Or, the control signal can be generated from a circuit internal to that nanopore structure. The control signal has the effect of changing the voltage level at each nanopore structure. The control signal can be applied via the electrical transduction element for modifying the voltage between the passage and the drive electrode(s). Additionally, or alternatively, the control signal can be applied via a further control electrode in the passage.

The device may have a single drive electrode provided in electrical connection with the analyte reservoir and a single drive electrode provided in electrical connection with the outlet chamber wherein the drive electrode serves to provide a common potential difference across the nanopore array.

Alternatively, the device may comprise a plurality of drive electrodes on the cis and/or the trans side of the device.

The application of a control signal to an individual nanopore structure functions to change the potential difference across the nanopore structure and between that nanopore structure and the drive electrodes. By way of example, the drive electrode in the analyte reservoir can have a voltage level of -<NUM> volts, while the drive electrode in the outlet chamber can have a voltage level of <NUM> volts such that the potential difference across the passages of the array is <NUM> volts. The application of a control signal to impose a voltage of -<NUM> volts at the nanopore structure results in a potential difference between the nanopore structure and the cis and trans electrodes or -<NUM> volts and -<NUM> volts, respectively.

The electrical transduction element and the control terminal of each nanopore can be directly connected. In doing so, the electrical transduction element can function as both a sensor electrode and a control electrode. This can be implemented by providing an electrical transduction element with two terminals: one for connecting to sensing circuitry, the other for connecting to control circuitry. In practice, the sensing circuitry and the control circuitry can reside in the same circuit or component. Any of the circuits can be located off-structure and connected to the structure via, for example, a wire-bond.

The control terminals can be configured to apply a control signal to alter the electrical potential difference from the drive electrodes to each respective nanopore structure in response to a measurement of the fluidic electrical potential at the electrical transduction element of that nanopore structure by said electrical transduction element. The application of the control signal is configured to alter the potential difference between at least one of the control terminals and at least one of the drive electrodes.

A control signal applied to the control terminal of a nanopore structure can alter the magnitude and/or the polarity of the potential difference between that nanopore and a drive electrode, which can change the rate at which an analyte passes though the passage of that nanopore structure or change the direction of movement of that analyte.

The control signal can be connectable to a plurality of the nanopore structures to simultaneously alter the potential difference between the connected control terminals and at least one of the drive electrodes.

The control signal can be applied for purposes other than to reject an analyte or control the speed and or direction of its translocation. For example, the control signal can be applied to induce insertion of a biological nanopore in a membrane supported by the nanopore structure. The electrical transduction elements can be connected to a measurement circuit to read signals received from the electrical transduction element. The nanopore structure can be provided with a switchable connection to a measurement circuit. Said switchable connection can disconnect the measurement circuit prior to the application of a control signal. In this way the control signal can be disconnected from measurement circuitry and inhibit the control signal influencing the performance of measurement circuitry.

In other words, the electrical transduction elements can be isolatable prior to the application of the control signal. Each individual electrical transduction element of each nanopore structure can be selectively isolated prior to application of the control signal.

The control signal can be applied for various purposes.

The control signal can be applied independently of measurements of the analyte. For example, the control signal can be applied to a membrane supported by the nanopore structure to induce insertion of a biological nanopore in the membrane.

The control signal can be applied to a nanopore structure in response to a measurement by the electrical transduction element.

By way of example, the control signal can be applied to for the purpose of unblocking a nanopore when the device determines that the passage through the nanopore is blocked, for example by analyte. The control signal can then be applied to unblock the passage.

The device is able to determine that the nanopore is blocked from the measurement of the change in electrical potential caused by the inhibition of current flow through the nanopore. In the absence of interaction of analyte with the nanopore, ion current flow through the nanopore due to the presence of an ionic salt in the aqueous sample may be referred to as the open pore current. When an analyte interacts with the nanopore, ion current flow through the pore is reduced and variation in the reduction in ion current may be measured as a fluctuation in electrical potential at the sensor electrode over time as an analyte such as DNA translocates the nanopore. A blockage of the nanopore, for example due to analyte becoming immobilised in the pore gives rise to a reduced ion current flow whose value changes very little over time. In a further example, the control signal can be applied to eject an analyte from the nanopore which is not of interest or which is no longer of interest. Measurements can be performed in real-time such that a decision to eject the analyte may be made before complete measurement of the analyte, for example a polynucleotide is made.

With regard to the prior mentioned devices for sequencing polynucleotides such as the MinION™ device, current flow though the nanopores is measured under the application of a potential difference between a respective array of electrodes provided on one side of each of the nanopores and a common electrode provided on the other side of the nanopores in an analyte reservoir. Because each nanopore has an associated electrode, it is possible to individually control the potential difference across each nanopore of the array and eject an analyte. In the hereinafter described embodiments, there are various advantages associated with carrying out measurement of the local potential at each nanopore by means of the electrical transduction elements. The drive electrodes serve to provide a potential difference across the nanopore array and not to measure analyte. Consequently, individual control of the potential difference at a nanopore by the drive electrodes is not possible. However, it is possible to provide individual control over the potential difference across each nanopore by means control terminals.

The array of nanopore structures can have circuits, each circuit associated with a respective nanopore structure and connected to the electrical transduction element. Each circuit can be configured to modify and/or process the signals received from the electrical transduction element. The circuit can also apply a control signal to the electrical transduction element. The circuit can isolate the control signal applied to the electrical transduction element from other sensing and processing functions.

Each circuit can reside within the pixel of the nanopore structure. Each circuit can be addressable. Each nanopore structure can be addressable. The addressing function can allow an external processor to communicate with a nanopore structure to at least one of receive measurement information or control movement of an analyte in the passage. In this way, the measurement and control of sensing at each individual passage can be independently controlled. The circuits may be provided on or embedded within the support structure.

Each electronic circuit can be associated with a group of nanopore structures. By way of example an electronic circuit can be shared by a group of four nanopore structures. Sensing and control of the nanopore structures in the group can be multiplexed. In this way the electronic circuit can be addressable, and multiplexing used to control individual nanopore structures.

Each circuit may be associated with a respective nanopore structure or a group of nanopore structures. Each circuit can be connected to the control terminal and/or the electrical transduction element, such that the circuit is configured to alter at the respective nanopore structure an electrical potential imposed by the drive electrodes in response to a measurement at the electrical transduction element and/or from an external processor attached thereto.

The structure has a nanopore layer incorporating an array of wells, each well having a membrane that spans the well and has a nanopore provided therein. The nanopores can be provided by a user after a device having nanopore structures has been provided to them. The nanopore layer can be replaced such that the device is recyclable. The nanopore structure also includes a base layer incorporating channels. The nanopore layer and the base layer are sandwiched together such that the nanopores, the wells and the channels are aligned to define the passages.

Each nanopore structure is defined by its passage. The passage fluidly connects the cis and trans sides.

The electrical transduction element defines a part of the passage. By way of example, the electrical transduction element can be sandwiched or laminated between the nanopore layer and the base layer. It can, however, be located elsewhere in the passage. It can be configured around the passage provided there is a direct fluid connection, between the electrical transduction element and a nanopore provided in the nanopore layer.

The electrical transduction element and/or the circuit can be implemented on a sense layer. The sense layer can be a sub-structure. The sense layer can be sandwiched or embedded between the nanopore layer and the base layer, said sense layer having a through-hole that aligns with the through-holes of the nanopore layer and the base layer. To be clear, the nanopore layer, sense layer and base layer can be sub-structures that are stacked to provide an array of nanopore structures.

A nanopore forms part of the passage. The rejection of an analyte can be managed using a control signal, which functions to control the movement of an analyte in the nanopore e.g. reject the analyte from the nanopore. The nanopore in a passage can become blocked. The blockage of a nanopore can be sensed and a control signal applied to the nanopore structure to clear the blockage.

The nanopore layer is formed with a well across which a membrane, such an amphiphilic membrane or a lipid bilayer can be formed such that a nanopore can be inserted in the membrane. One nanopore is provided for each nanopore structure in the array.

In a further aspect, the invention provides a method as defined in claim <NUM>.

Fluidic electrical potential can be measured at the electrical transduction element. The fluidic electrical distribution across that nanopore structure can be altered when the device is provided with a fluid. In operation, a fluid resides in the reservoir, chamber and passages of the nanopore structure. The fluid in the reservoir, chamber and passages of the nanopore structure can be different fluids.

The electrical potential difference imposed across the array serves to induce an analyte through, or at least in to, the passage. An analyte to be analysed is provided in the analyte reservoir and induced to the outlet chamber, which is achieved by the drive electrodes. The situation can, however, be reversed in that an analyte can be provided in the outlet chamber or an analyte in the outlet can be induced by the drive electrode in to the analyte reservoir e.g. by changing the potential difference between the drive electrodes.

In each case, the electrical transduction elements of each nanopore structure, which are provided with nanopores to function as nanopore sensors, can measure a change in the fluidic electrical potential. The array of nanopore structures is dimensioned such that the electrical transduction element of one nanopore sensors is inhibited from detecting an analyte passing through a nanopore in a neighbouring nanopore structure.

A control signal is applied to an element to alter the electrical potential difference across the nanopore sensor in which said element resides.

The control terminal connected to the electrical transduction element can be switchably connected to the control terminal of the electrical transduction element for applying the control signal thereto. Additionally, or alternatively, the device can be operated to isolate any sensing circuitry from the electrical transduction element to inhibit damage to said circuitry while the control signal is applied.

The method can include analysing characteristics of the change in the electrical potential locally at a nanopore sensor and applying the control signal to that nanopore sensor in response to predetermined characteristics. The method applies a control signal to an electrical transduction element of a nanopore sensor to alter the potential difference imposed by the drive electrodes at that nanopore sensor. The change in potential difference can induce movement of an analyte or a free-moving nanopore, which can be charged.

The control signal can perform a plurality of operations including, but not limited to: inducing pore insertion in to a membrane formed across the passage; unblocking a nanopore; rejecting an analyte; altering the rate of translocation of an analyte through that nanopore.

Embodiments of the invention are discussed below, by way of non-limiative example only, with reference to the drawings in which:.

The embodiments include corresponding components, typically labelled by a common reference numeral. For purposes of clarity, the description of corresponding components is not repeated but applies generally to all embodiments, except where the context demands otherwise. Not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to understand the invention.

<FIG> are sectional views of a portion of a structure <NUM> having a nanopore structure incorporated therein. The structure <NUM> has an array of nanopore structures, each nanopore structure adapted to support a nanopore <NUM>. The nanopore structures of the device can function as nanopore sensors when configured with a nanopore. A nanopore sensor <NUM> herein is nanopore structure having a nanopore.

<FIG> illustrate that a plurality of the nanopore sensors <NUM> shown in <FIG> is arranged as part of an array of nanopore structures <NUM>. Such an arrangement may be referred to as a two-dimensional matrix of nanopore structures, or an array of nanopore sensors.

The structure <NUM>, which may take the form of a sheet, incorporates the array of nanopore structures <NUM> (n. only one nanopore sensor <NUM> of the array is shown) and can be configured within a device or device for analysing an analyte, as shown in <FIG>.

The structure <NUM> separates the analyte reservoir <NUM> for receiving an analyte and an outlet chamber <NUM>. The structure <NUM> has a nanopore layer <NUM> configured upon a base layer <NUM>, which together forms at least a portion of the structure <NUM> having a plurality of nanopore sensors <NUM>. Each nanopore sensor <NUM> in the array <NUM> has a passage <NUM>, or fluidic passage, configured to extend through the nanopore layer <NUM> and base layer <NUM> of the array <NUM> for connecting the analyte reservoir <NUM> and outlet chamber <NUM>. The analyte reservoir <NUM> may also be known as an analyte chamber, a sample chamber, a cis, a cis reservoir, or a first fluidic reservoir. The outlet chamber may also be known as a trans, a trans reservoir, or a second fluidic reservoir.

The nanopore layer <NUM> of each nanopore sensor <NUM> is provided with a nanopore <NUM> in a membrane <NUM> supported by the nanopore layer <NUM>. The nanopore <NUM> is provided in a membrane <NUM> proximal the first end <NUM>, or pore end, of the passage <NUM> (e.g. at the top of the sensor as shown).

The base layer <NUM> has a channel <NUM> proximal a second end <NUM>, or channel end, being an opposite end of the passage <NUM> to the first end <NUM> (e.g. at the bottom of the nanopore sensor <NUM> as shown). The passage <NUM> extends through the nanopore structure <NUM> connecting one side to the other. The channel <NUM> forms part of the passage <NUM>. The channel <NUM> is structurally and geometrically configured to function as a fluidic resistor. This can be achieved by defining the aspect ratio of the channel <NUM>. Additionally, or alternatively other techniques for implementing fluidic resistance in the channel <NUM> can be used.

The fluidic resistance of the channel <NUM> can be varied by varying its dimensions, in particular its aspect ratio and by varying the ionic concentrations of the fluids in the analyte reservoir <NUM> and the outlet chamber <NUM>. For example, the channel <NUM> can be configured with a high aspect ratio to increase the resistance. Additionally, or alternatively, the fluid in the channel <NUM> can have a lower ionic concentration compared to the fluid in the analyte reservoir <NUM> and outlet chamber <NUM> to increase the channel's resistance. Maintaining a higher ionic concentration in the analyte reservoir <NUM> and the outlet chamber <NUM> improves the signal to noise ratio.

In some embodiments, the aspect ratio may, for example, be between about <NUM>:<NUM> to about <NUM>:<NUM> , which is a ratio of channel length to channel diameter or largest transverse dimension.

In some embodiments, the ionic concentration difference may be between about <NUM>:<NUM> to about <NUM>:<NUM>, for example around <NUM>:<NUM>, which is a ratio of ionic concentration in the analyte reservoir <NUM> and/or outlet chamber <NUM> to the ionic concentration in the channel <NUM>.

The channel <NUM> can be configured such that the resistance of the channel <NUM> and the nanopore <NUM> are substantially matched, when the passage <NUM> is occupied by fluid, and relatively high relative to the resistance of fluid in the analyte reservoir <NUM> and outlet chamber <NUM> such that the resistance of the analyte reservoir <NUM> and outlet chamber <NUM> does not appreciably influence the measurements. In other words, the channel <NUM> is configured as a fluidic resistor to approximate the resistance of the nanopore <NUM>. This means that the resistance of other circuit elements such as the fluid in the analyte reservoir <NUM> and outlet chamber <NUM> has less significance and does not require compensation to account for it when measurements are taken.

The signal-to-noise ratio may be optimised by selecting the fluidic resistance of the channel <NUM> to be equal to the resistance of the nanopore <NUM>. However, this is not essential and the fluidic resistance of the channel <NUM> may be varied from this value to take account of other factors, while still obtaining an acceptable signal-to-noise ratio. An acceptable signal-to-noise ratio may be achieved with the fluidic resistance of the channel <NUM> being significantly less than the resistance of the nanopore <NUM>, for example with the fluidic resistance of the channel <NUM> being <NUM>% or less of the resistance of the nanopore <NUM>, for example <NUM>% of the resistance of the nanopore <NUM> or less. In some embodiments, a lower limit on the fluidic resistance of the channel <NUM> may be set by the desired signal to noise ratio. In other embodiments, a lower limit on the fluidic resistance of the channel <NUM> may be set by the threshold for crosstalk between adjacent channels during flicking (as described below). That is, the fluidic resistance of the channel <NUM> is desirably significantly greater than the resistance from the end of the channel <NUM> to the electrical transduction element to prevent these resistances forming a voltage divider which applies a fraction of the applied voltage to adjacent channels <NUM>.

Other factors that may be considered in the selection of the fluidic resistance of the channel <NUM> are as follows.

As the fluidic resistance of the channel <NUM> increases, the diffusion of ions decreases, causing increased depletion of ions near the pore, and thereby causing a decay of the signal over the timescale of a typical event over which a signal is obtained. In order to increase the limit on read length caused by this effect, the fluidic resistance of the channel <NUM> may be reduced. In many embodiments, this factor may place an upper limit on the fluidic resistance of the channel <NUM>.

As the channel <NUM> and the nanopore <NUM> act as a voltage divider, the voltage across the nanopore <NUM> is affected by the current flowing through it. As the fluidic resistance of the channel <NUM> increases, the variation in the voltage across the nanopore <NUM> increases, which can complicate signal processing. In order to limit this effect, the fluidic resistance of the channel <NUM> may be reduced.

Channels having lower fluidic resistances are easier to fabricate, and may open up alternative fabrication techniques that improve yield or reduce cost.

Reducing the fluidic resistance of the channel <NUM> may increase bandwidth or provide leeway for additional capacitance in the passage <NUM>.

Taking into account these factors, the fluidic resistance of the channel <NUM> may be less than the resistance of the nanopore <NUM>, typically at most <NUM>%, or at most <NUM>% of the resistance of the nanopore <NUM>. In some embodiments, the optimal fluidic resistance of the channel <NUM> may be around <NUM>% of the resistance of the nanopore <NUM>.

When reducing the ratio of the fluidic resistance of the channel <NUM> to the resistance of the nanopore <NUM>, the signal to noise ratio does not scale directly with that resistance ratio. For example, in some embodiments when the fluidic resistance of the channel <NUM> is around <NUM>% of the resistance of the nanopore <NUM>, then the signal to noise ratio is around <NUM>% of its optimal value.

The channel <NUM> can be formed in a wafer, and after a passage <NUM> is formed therethrough an oxide layer can be used to reduce the diameter of the passage <NUM> through the base layer <NUM>, thus enabling the amount of oxidisation to adjust the aspect ratio.

A sensor electrode <NUM>, or sensor element, is disposed between the nanopore <NUM> and at least a portion of the channel <NUM>. The sensor electrode <NUM> forms the electrical transduction element in this example. More generally, the sensor electrode <NUM> could be adapted to form an electrical transduction element of any of the various types disclosed in <CIT>.

The sensor electrode <NUM> is exposed, at least in part, to the passage <NUM> in the nanopore sensor <NUM>, and configured with a connection <NUM> for measuring electrical potential of the fluid at the location of the sensor electrode <NUM> when a fluid is provided in the passage <NUM>. Together with the nanopore layer <NUM> and base layer <NUM>, the sensor electrode <NUM> defines the walls of the passage <NUM>. The connection <NUM> can be a wire-bond to a separate electronic circuit <NUM>, said electronic circuit <NUM> configured to analyse signals obtained from the sensor electrode <NUM>.

The analyte reservoir <NUM> can function as a first fluidic reservoir, while the outlet chamber <NUM> can function as a second fluidic reservoir. The structure <NUM> can separate, at least in part, the analyte reservoir <NUM> and the outlet chamber <NUM> and the passage <NUM> of a sensor <NUM> connects the analyte reservoir <NUM> to the outlet chamber <NUM>.

In use, the passage <NUM> of each nanopore sensor <NUM> is occupied by a fluid. Further, drive electrodes <NUM> in the analyte reservoir <NUM> and outlet chamber <NUM> comprise at least one respective cis electrode 132a, also known as an analyte electrode, and at least one respective trans electrode 132b, also known as an outlet electrode, configured to impose an electrical potential difference across the passages <NUM> of the nanopore sensors <NUM> in the array <NUM> between the analyte reservoir <NUM> and the outlet chamber <NUM>.

The structure <NUM> can be substantially planar. The array <NUM> can be substantially planar. Non-planar configurations are envisaged by the inventors but not described herein. The nanopore sensors <NUM> in the array <NUM> have a cis-surface <NUM> of the nanopore layer <NUM> arranged facing the analyte reservoir <NUM> and defining a cis-plane <NUM>, and a trans-surface <NUM> of the base layer <NUM> for facing the outlet chamber <NUM> and defining a trans-plane <NUM>. This cis-plane <NUM> and trans-plane <NUM> are indicated by the hashed line in <FIG>, <FIG> and <FIG>. The sensor electrode <NUM> is embedded within the structure <NUM> between the cis-plane <NUM> and the trans-plane <NUM>. The nanopore <NUM> can lie substantially on the cis-plane <NUM> at the first end <NUM> of the passage <NUM> while the second end <NUM> of the passage <NUM> can lie substantially on the trans-plane <NUM>.

As shown in the assembly of <FIG>, the sensor electrode <NUM> can, at least in part, be embedded between in the structure <NUM> between the nanopore layer <NUM> and the base layer <NUM>. In other words, the sensor electrode <NUM> is sandwiched or laminated between the nanopore layer <NUM> and the base layer <NUM>.

The nanopore layer <NUM> has a well <NUM> formed at the first end <NUM> of the passage <NUM>. In the example of <FIG> the nanopore <NUM> is configured at the first end <NUM> of the passage <NUM>, on one side of the well <NUM>, substantially on the cis-plane <NUM>. The sensor electrode <NUM> is configured on the opposite side of the well <NUM> to the nanopore <NUM>, as shown. The well <NUM> is shown as a cup-shaped recess with a membrane <NUM>, shown in cross-section, across its rim. The well <NUM> is configured to receive an analyte that has passed through a nanopore <NUM>. Note that the well <NUM> is fluidly connected to the channel <NUM> via a well aperture 142a, which can be described as a well outlet. The well aperture 142a functions to enable the analyte chamber to be fluidly connected to the outlet chamber. The well aperture 142a does not function as a nanopore. In some implementations, the well aperture 142a is configured to enable an analyte to pass therethrough uninhibited i.e. without influencing movement of the analyte from the analyte reservoir <NUM> to the outlet chamber <NUM>.

Although the well aperture 142a provides a fluid connection between the analyte reservoir <NUM> and outlet chamber <NUM> an analyte that has passed through the nanopore <NUM> can remain in the well <NUM>. The well <NUM> and channel <NUM> can be considered part of the outlet chamber <NUM>. In the example shown in <FIG>, the well aperture 142a is centrally located at the base of the well <NUM> within the sensor electrode <NUM>.

The well <NUM>, and more generally the nanopore layer <NUM>, is configured for supporting a membrane <NUM> such as a polymer membrane or lipid bilayer, which may be referred to as a fluid membrane. The nanopore layer <NUM> can be fabricated separately from the base layer <NUM>. The nanopore layer <NUM> can be formed from a different material from the base layer <NUM>. The nanopore layer <NUM> can be formed from at least one of: a photolithographically prepared material; a moulded polymer; or a laser etched plastic.

In some embodiments, the sensor electrode <NUM> is directly connectable to the base or gate of a sensing transistor <NUM> (shown in <FIG> and described further below) for measuring electrical potential of the fluid at the location of the sensor electrode <NUM> when a fluid is provided in the passage <NUM>. In some cases, the sensor electrode <NUM> can be connected to an edge-connector or wire-bond, optionally by a conductive via and/or interconnect, to an electronic circuit <NUM>, which is an example of a measurement circuit, off-structure. The sensing transistor <NUM> can be a field effect transistor (FET) and the configuration of the sensing transistor <NUM> and its optional integration in to the structure <NUM> is described in an example below. In some embodiments, the sensing transistor <NUM> (not shown) can be located in the electronic circuit <NUM>.

The nanopore sensor <NUM> shown in <FIG> is an example in which the sensor electrode <NUM> can be formed upon the base layer <NUM>. While the sensor electrode <NUM> of <FIG> can be formed on the base layer <NUM> directly it can, alternatively, be formed separately upon a sense layer <NUM> , as depicted in <FIG> and <FIG>. After forming the sensor electrode <NUM> on a sense layer <NUM>, the sense layer <NUM> is then sandwiched between the nanopore layer <NUM> and base layer <NUM>, resulting in the structure shown in <FIG>.

The sense layer <NUM> can be fabricated in a similar manner to the base layer <NUM>, wherein a wafer has passages <NUM> formed therethrough, substantially perpendicularly to the surfaces of the wafer. Alternatively, the wafer can be post-processed to open up the passage <NUM>. The passages <NUM> and/or channels <NUM> can be formed using techniques such as photolithography or deep reactive-ion etching (DRIE) or combinations thereof. The wafer can be enclosed by an oxide layer. The wafer can be a CMOS wafer. The sensor electrode <NUM> can be formed on the sense layer <NUM> around the passages <NUM> on one side of the sense layer <NUM>. The passages <NUM> through the sense layer <NUM>, and the sensor electrode <NUM> formed around these passages <NUM>, are arranged to have a pitch or layout that results in alignment with channels <NUM> on the base layer <NUM>. When secured together, the passages <NUM> of the sense layer <NUM> are aligned with the channels <NUM> of the base layer <NUM>.

By way of example, the nanopore layer <NUM> may be made of polymer, which can be moulded or lithographically etched; the base layer <NUM> may be formed of a silicon wafer; and/or the sense layer <NUM> may be a CMOS wafer.

The sense layer <NUM> can be aligned and bonded to the base layer <NUM> in one of two orientations. In one orientation (not shown) the sensor electrode <NUM> remains fully exposed after bonding - that is to say that the sensor electrode <NUM>: is not sandwiched between the sense layer <NUM>; is distal from the base layer <NUM> after the sense layer <NUM> is secured to the base layer <NUM>; and is subsequently sandwiched between the sense layer <NUM> and the nanopore layer <NUM>. In the other orientation, as shown in <FIG>, the sensor electrode <NUM> is formed on top of a sense layer <NUM> which is then inverted before bonding to the base layer <NUM> such that the sensor electrode <NUM> faces down, as viewed, and is sandwiched between the sense layer <NUM> and the base layer <NUM>. Prior to bonding in this configuration, a section of the oxide layer on the base layer <NUM> around the channel <NUM> can be etched away or otherwise removed to create a cavity <NUM> such that there is an increased area of the sensor electrode <NUM> exposed to the passage <NUM> after bonding. The area of exposed sensor electrode <NUM> can be maximised to increase contact with a fluid in the passage <NUM>.

The wells <NUM> of the nanopore layer <NUM> are aligned with the passages <NUM> and sensor electrodes <NUM> and bonded to the sense layer <NUM> with the sensor electrodes <NUM> sandwiched therebetween. Looking at <FIG>, and noting that the sense layer <NUM> is fabricated from the bottom upwards, the last stage is the application of the sensor electrode <NUM> on top. When assembled, the sense layer <NUM> is flipped over such that the sensor electrode <NUM> that was on top is now facing downwards, as shown in <FIG>. The cavity <NUM> etched out of the base layer <NUM> oxide layer (the grey part) means that the sensor electrode <NUM> is sufficiently exposed.

The sensor electrode <NUM> remains exposed, at least in part, to the passage <NUM> and configured with a connection for measuring electrical potential of the fluid at the location of the sensor electrode <NUM> at the nanopore <NUM> when a fluid is provided in the passage <NUM>. Arrangements of the sensor electrode <NUM> - which minimise its surface area openly facing one of the analyte reservoir <NUM> or outlet chamber <NUM> (e.g. the arrangements of <FIG>) - function to limit exposure to the analyte reservoir <NUM> or outlet chamber <NUM> to inhibit contamination of the surface of the sensor electrode <NUM>. One such example is shown in <FIG> that shows the sensor electrode <NUM> substantially partially enclosed in the passage <NUM>. Before population with a fluid, or during the formation of an amphiphilic membrane for supporting a biological nanopore, the surface of the sensor electrode <NUM> can be exposed to fluids that could contaminate the surface of the sensor electrode <NUM>, thus if there is a contamination risk then it can be mitigated.

In one configuration, at least a portion of the sensor electrode <NUM> can be arranged to face away from the well <NUM> toward the channel <NUM>, as shown in <FIG>. An exposed portion of the sensor electrode <NUM> provides a connection to a fluid in the passage <NUM> for sensing fluctuations in the fluidic electrical potential at the sensor electrode <NUM> when an analyte passes through the nanopore <NUM>. The sensor electrode <NUM> may also have an embedded portion embedded within the structure <NUM>. The sensor electrode <NUM> can also have a connection <NUM>, such as a wire-bond, for connection to an electronic circuit <NUM>, such as a measurement circuit or control circuit, which can be separate from the structure <NUM> as shown in <FIG>.

In each of the examples, the sensor electrode <NUM> can be configured in various configurations for exposure to a fluid within the passage <NUM> and can, at least one of: cover, at least in part, a wall of the passage <NUM>; cover, in cross-section, a portion of a wall of the passage <NUM>; form an annulus around the passage <NUM>; form, at least in part, the surface of the base layer <NUM> or the sense layer <NUM> around the passage <NUM> and have an exposed portion arranged to face the analyte reservoir <NUM>; form, at least in part, the surface of the sense layer <NUM> around the passage <NUM> and have an exposed portion arranged to face the outlet chamber <NUM>. In particular, a cavity <NUM> can be formed in the passage <NUM> to create a region that enables the area of sensor electrode <NUM> exposure to be increased and come in to contact with an increased amount of fluid. The cavity <NUM> is formed by recesses formed in the base layer <NUM> and/or sense layer <NUM> before the base layer <NUM> and sense layer <NUM> are aligned and connected.

While the sensor electrode <NUM> can have a minimal degree of exposure to the fluid in a well, such as in the form of a nanowire, the inventors have proposed the examples herein to optimise performance of the nanopore sensor <NUM> and improve manufacturability.

As shown in <FIG> the sensor electrode <NUM> is substantially planar and shaped to accommodate the passage <NUM>. In other words, the sensor electrode <NUM> is configured to enable uninhibited fluid communication between the analyte reservoir <NUM> and outlet chamber <NUM>, which can be achieved by either (i) shaping the sensor electrode <NUM> to extend around the passage <NUM> or well aperture 142a, and/or (ii) forming a sensor aperture <NUM> in the sensor electrode <NUM>.

The footprint of the exposed portion of the sensor electrode <NUM> can be any shape. The well <NUM> of <FIG> and the cavity <NUM> of <FIG> can be cylindrical such that the floor of the well <NUM> is circular, or a planar surface of the cavity <NUM> is curved. These configurations result in the exposed portion of the sensor electrode <NUM> being circular or disc-shaped. In the examples shown a sensor aperture <NUM> is provided such that the footprint of the exposed portion is shaped like an annulus. The exposed area of the sensor electrode <NUM> can be maximised, which can mean covering at least one face or surface of the well <NUM> and/or cavity <NUM>.

The sensor electrode <NUM> and the sensor aperture <NUM> are shown as circular but could have other shapes. In some embodiments having circular shapes, the ratio of the radius of the exposed portion of the sensor electrode <NUM> to the radius of the sensor aperture <NUM> can be in a range from <NUM>:<NUM> to <NUM>:<NUM> or about <NUM>:<NUM>. In the case of non-circular shapes, the ratio of the square roots of the areas may have the same values.

Alternatively, the area of exposed portion of the sensor electrode <NUM> can be expressed in relation to the ratio between the area or footprint as viewed of the sensor aperture <NUM> can be about <NUM>:<NUM>, or about <NUM>:<NUM> or about <NUM>:<NUM>.

By way of example, the sensor electrode <NUM> may have a diameter (or maximum dimension) in a range from <NUM> to <NUM> and the sensor aperture <NUM> may have a diameter (or maximum dimension) in a range upwards from <NUM>. The sensor aperture <NUM> does not function as a sensor so its size does not have an upper limit within the bound that it is desirable to minimise the restriction of the remaining area of the sensor electrode <NUM>.

The sensor electrode <NUM> may be formed from a suitable conductive material, for example platinum or gold.

While <FIG> have a sensor electrode <NUM> having a connection <NUM> to a separate electronic circuit <NUM>, <FIG> illustrates that the structure <NUM> and array <NUM> can accommodate an integrated circuit <NUM>. The integrated circuit <NUM> can incorporate one or more of the functions of the electronic circuit <NUM>. In other words, various functions, such as sensing, amplifying, controlling, filtering, reading out etc which can be implemented on the separate electronic circuit <NUM> can be implemented, alternatively, on the integrated circuit <NUM>. The integrated circuit <NUM> can be formed on a separate layer or wafer and subsequently connected to the sense layer <NUM> having the sensor electrode <NUM> thereon. The inventors envisage, however, that the integrated circuit <NUM> is fabricated within the sense layer <NUM> together with the sensor electrode <NUM>. An integrated circuit <NUM> can be provided for each nanopore sensor <NUM>.

In some approaches, after fabrication of the sense layer <NUM> having the integrated circuit <NUM> and sensor electrode <NUM> formed and exposed on one side, the sensing structure is flipped and bonded to the base layer in the same way as it was in relation to <FIG>. Connections <NUM> (not shown in <FIG> connect the integrated circuit <NUM> with a connector <NUM> for sending signals or data produced by the integrated circuit <NUM> off the structure. The connections <NUM> can be connected to a connector <NUM> as shown in <FIG>, although other configurations are implementable. With the sense layer <NUM> connected to the base layer <NUM> the nanopore layer <NUM> can be formed thereon such that the sense layer <NUM> is sandwiched between the nanopore layer <NUM> and the base layer <NUM>. As before with <FIG>, when bonded together, the passages of the sense layer <NUM> align with the channels of the base layer <NUM> and the well <NUM> of the nanopore layer <NUM> form a portion of the passage <NUM>.

In use, the electronic circuit <NUM> and/or integrated circuit <NUM> is configured to detect resistance changes at the nanopore <NUM> when an analyte, such as a polymer, passes through the nanopore <NUM>, said resistance change detected through the fluid in the nanopore sensor <NUM> (strictly speaking a measure of resistance being detected as a voltage over the effective voltage divider, as described above). In an array <NUM> the integrated circuit <NUM> of each nanopore sensor <NUM> can be communicably addressable. In light of parasitics, noise from communications and background noise the detected resistance can be difficult to read directly using an off-board processor. To provide a processor with a better signal, i.e. a cleaner reduced noise signal, the integrated circuit <NUM> can be configured to locally transform or modify or otherwise process signals derived from the detection of a polynucleotide or other analyte passing through the nanopore <NUM>. The integrated circuit <NUM> can be configured to at least one of: amplify signals, such as amplifying a voltage level of the signal; filter the signal, for example to remove noise; sample the signal; digitise the signal using an analogue to digital converter (ADC) implemented in the electronic circuit <NUM>.

The integrated circuit <NUM> can be formed within a nanopore sensor <NUM> footprint within the array <NUM> of the structure <NUM>.

By way of example, each nanopore sensor <NUM> of the array <NUM> can be contained within a pixel <NUM> which is a footprint of the nanopore sensor <NUM>, as viewed in <FIG>, which can be considered to represent a schematic plan view of a nanopore sensor <NUM> shown in <FIG>. As viewed, each pixel <NUM> accommodates a nanopore sensor <NUM>, an sensor electrode <NUM> and integrated circuit <NUM>. The sensor electrode <NUM> and integrated circuit <NUM> can be arranged to inhibit noise interference created by the integrated circuit <NUM> from being detected by the sensor electrode <NUM>. For example, the integrated circuit <NUM> may be separated from the nanopore sensor <NUM>, as depicted in <FIG>. This separation can be implemented by configuring the integrated circuit <NUM> to be located outside the pixel <NUM>, as viewed.

This separation may also simplify the manufacturing process. Alternatively, the integrated circuit <NUM> can be distanced from the sensor electrode <NUM> (i.e. the distance between the sensor electrode <NUM> and the integrated circuit <NUM> in the depth direction, or thickness of the structure <NUM>, is increased to minimise noise interference. Note that the depth direction of <FIG> is in a direction in to and out of the page, as viewed.

In the example shown, the pixel <NUM> is square and has a side length of <NUM>, but in other examples may be in a range from <NUM> to <NUM>.

By way of example, the integrated circuit <NUM> occupies about three-quarters of the pixel <NUM>, while the remaining quarter is occupied by the sensor electrode <NUM> which has a diameter of <NUM> in the example shown.

Other arrangements are envisaged. In some embodiments, the sensor electrode <NUM> may be larger than the example shown in <FIG>, for example covering almost all of the area of the nanopore sensor <NUM>. In some embodiments, the sensor electrode <NUM> may be have other shapes covering more area, for example square or rectangular. The sensor electrode <NUM> may have dimensions of up to <NUM>, in which case it may have an area of up to <NUM><NUM>, depending on its shape.

For packaging efficiency, the pixel <NUM> can be tessellated, and, for example, the tessellation can be hexagonal.

Each nanopore sensor <NUM> has a passage <NUM>, although during fabrication of the base layer <NUM> more channels <NUM> could be created in the base layer <NUM> than are needed, depending on the method of fabrication. Some methods of fabrication such as reactive ion etching can etch a single channel <NUM> for each pixel <NUM>. Some other method such as photo assisted electrochemical etching requires a high-density array of channels <NUM> to be etched at the same time to maintain the geometry of those channels <NUM> - in this case unused channels <NUM> in the base layer <NUM> are blocked during fabrication of the array <NUM> such that only one channel <NUM> and one passage <NUM> are provided per pixel <NUM>. The density of the channels <NUM> formed in the base layer <NUM> can vary. <FIG> shows, by way of comparison, a cross-section of a nanopore sensor <NUM> having a lower density of blocked channels 122a than that shown in <FIG>. The channels <NUM>, as shown in <FIG> can be blocked prior to the sense layer <NUM> being added to the base layer <NUM>, or may be blocked by a substrate of the sense layer <NUM>. It is to be noted that <FIG> is shown with portion of two nanopore sensor <NUM>, each with its own passage <NUM>, and has not yet had a nanopore layer <NUM> added upon the sense layer <NUM>.

<FIG> shows the nanopore sensor <NUM> pixel <NUM> of <FIG> arranged in a 6x6 layout providing an array <NUM> of <NUM> nanopore sensors <NUM>, while <FIG> has an 18x18 array having <NUM> nanopore sensors <NUM>. The array size can be 1000x1000, providing <NUM>,<NUM>,<NUM> nanopore sensors <NUM>. In the present example, an array of one million nanopore sensors <NUM> of the type shown in <FIG> would have a footprint of <NUM><NUM>, however nanopore sensors <NUM> having pixels <NUM> as small as <NUM> can bring the footprint of a one million nanopore sensor array <NUM> down to around <NUM><NUM>. The array size can be <NUM>,<NUM>. The array <NUM> may comprise any number of nanopore sensors <NUM> between <NUM> and <NUM> million nanopore sensors <NUM>.

<FIG> shows an array <NUM> having nanopore sensors <NUM> as described herein arranged in a structure <NUM> provided in a device <NUM> for receiving and analysing an analyte of polymer such as nucleic acid. The device <NUM> may also be known as a sensor device or measurement system. The array <NUM> can be a sub-component of the device <NUM>. The array <NUM> can be a disposable component and replacable. Additionally, or alternatively, the nanopore layer <NUM> of the array <NUM> can be a disposable component and replacable. The device <NUM> can include an electronic circuit <NUM> as described above.

In some embodiments, processing of the signals measured by a nanopore sensor <NUM> can be performed by the electronic circuit <NUM>. The integrated circuit <NUM> can perform pre-processing prior to further analysis by the electronic circuit <NUM> of the device <NUM>.

In some embodiments, the device <NUM> houses the structure <NUM> to separate and define the analyte reservoir <NUM> and outlet chamber <NUM>. While often referred to, respectively, as the cis and the trans, the analyte can flow from the analyte reservoir <NUM> to the outlet chamber <NUM>. The array <NUM> has a plurality of nanopore sensors <NUM>, each with a passage <NUM> therethrough, to fluidly connect the analyte reservoir <NUM> and outlet chamber <NUM>. By way of example, the drive electrodes <NUM> in the analyte reservoir <NUM> and outlet chamber <NUM> can impose an electrical potential difference across the passage <NUM>, between the analyte reservoir <NUM> and outlet chamber <NUM>, to induce an analyte to flow from the analyte reservoir <NUM> to the outlet chamber <NUM>. The drive electrodes <NUM> can be configured such that the potential difference is substantially the same across all the nanopore sensors <NUM>.

Additionally, or alternatively, the device <NUM> can be configured to induce an analyte from the analyte reservoir <NUM> to the outlet chamber <NUM> using other techniques. As an analyte passes through a nanopore <NUM> the fluctuation in electrical potential caused by changes in ion current flow is detected by the sensor electrode <NUM>.

The sensor electrode <NUM> can function as, or be connected directly to, the base or gate of a sensing transistor <NUM> (as shown in <FIG> and described further below), which can be a field effect transistor (FET) device for example. The sensing transistor <NUM> outputs a signal that can be processed by the integrated circuit <NUM> of each nanopore sensor <NUM>, which can then be addressed in a row-column type manner. For example, the voltage at the drain of the sensing transistor <NUM> may depend upon the electrical potential sensed by the sensor electrode <NUM>, and the voltage at the drain can be read out, along with other drain voltages on other nanopore sensors <NUM> in an array <NUM>, in a row-column manner. The processed signal can then be analysed further - off the array <NUM> - to determine one or more properties of the analyte.

In the examples above each pixel <NUM> has its own integrated circuit <NUM>, but an integrated circuit <NUM> can be configured to serve a plurality of nanopore sensors <NUM>. In <FIG>, four nanopore sensors <NUM> are shown as a sensor module 102a (which may be part of a larger array of nanopore sensors <NUM>), wherein the integrated circuit <NUM> is common to four centrally located sensor electrodes <NUM>, as shown. Other configurations are feasible. In such module configurations the information or data obtained from each individual nanopore sensor <NUM> is addressable for control and/or retrieval of information. While the examples above have a dedicated integrated circuit <NUM> for each nanopore sensor <NUM>, combining nanopore sensors <NUM> in a sensor module 102a enables the efficiency of the layout to be improved. Efficiency improvements can, for example, be achieved because a common filter is used for each of the nanopores <NUM> within the sensor module 102a. This is possible if the integrated circuit <NUM> switches or multiplexes between the individual nanopore sensors <NUM> in turn. By sharing functions between the nanopore sensors <NUM> either the footprint of the integrated circuit <NUM> can be reduced or, alternatively, more functions can be accommodated.

<FIG> is a schematic representation of the immediate connections to the sensor electrode <NUM> with each nanopore sensor <NUM> in the array <NUM>. The cis electrode 132a can be connected to ground while a translocation voltage is applied to trans electrode 132b. The resistance of the nanopore <NUM> and the resistance of the channel <NUM>, which is configured to function as a fluidic resistor, dominate the circuit between the cis electrode 132a and trans electrode 132b via each passage <NUM> of each nanopore sensor <NUM>. In this way the circuit behaves like a voltage divider having two resistors of similar value. The nanopore resistance and resistance of the channel <NUM> or fluidic resistor, are approximately the same such that an electrode positioned therebetween is optimally positioned to detect changes in the nanopore resistance caused by an analyte passing therethrough. The sensor electrode <NUM> resides, as described above, in the region of each nanopore <NUM>. The sensor electrode <NUM> can lie between the nanopore <NUM> and the channel <NUM>. The effective impedance of the nanopore <NUM> and the channel <NUM> are much larger than the bulk fluidic resistance of the analyte reservoir <NUM> and outlet chamber <NUM> - this means that <FIG> can be used to model the circuit between the cis electrode 132a and trans electrode 132b.

The circuitry includes a sensing circuit <NUM> which measures fluidic electrical potential at the sensor electrode <NUM> of the nanopore sensor <NUM> for taking measurements from the nanopore <NUM>. The sensing circuit <NUM> may include, for example a sensing transistor <NUM> which may be a field effect transistor (FET). In this case, the sensor electrode <NUM> may be connected to the base of the sensing transistor <NUM>. The sensing circuit <NUM> may reside, at least in part, in the integrated circuit <NUM>. Thus, the sensor electrode <NUM> may be connected to a sensor terminal <NUM> of the sensing circuit <NUM>, as shown in <FIG>.

The sensing circuit <NUM> includes a control circuit <NUM> which applies a signal to the sensor electrode <NUM> to alter an electrical potential difference across the nanopore <NUM> imposed by the drive electrodes <NUM> in response to a control signal. The control circuit <NUM> includes a control terminal <NUM> which may be a field effect transistor (FET). In this case, the sensor electrode <NUM> is connected to the drain of the control terminal <NUM>. The control circuit <NUM> can reside, at least in part, in the integrated circuit <NUM>. Thus, the sensor electrode <NUM> is connected to a control terminal <NUM> of the control circuit <NUM>, as shown in <FIG> for application of the control signal.

The application of the control signal enables an alteration of the potential difference imposed across the individual nanopore <NUM> by altering the potential difference between the control connection of the control circuit <NUM> and the cis electrode 132a and/or the trans electrode 132b. The signal applied to the sensor electrode <NUM> can be a reverse-voltage that induces the charged analyte, such as a species, to change the direction in which it is moving through the passage <NUM>.

In some cases, the voltage applied can be an alternating current voltage, although other voltage waveforms (e.g., ramp, step, impulse, DC) may alternatively be applied.

The integrated circuit <NUM> of <FIG> enables a common electrode to be configured for each of the analyte reservoir <NUM> and outlet chamber <NUM>, while each nanopore sensor <NUM> can operate to detect an interruption to ion current flow across the passage <NUM> by detecting variations in electrical potential caused by a variation in nanopore resistance. Furthermore, the integrated circuit <NUM> enables each nanopore sensor <NUM> within the array <NUM> to be individually controlled to enable the sensor electrode <NUM> to either detect an analyte passing through the nanopore <NUM> through, for example, a connection with a sensing transistor <NUM> or control the flow of a charged analyte, such as a species, in the passage <NUM> of individual nanopore sensors <NUM> in the array <NUM> by adjusting the voltage applied to the sensor electrode <NUM> using control transistor <NUM>. The control of the flow of a charged analyte, such as a species, in the passage <NUM> of individual nanopore sensors <NUM> in the array <NUM> allows for an analyte passing through the nanopore <NUM>, or an analyte blocking the nanopore <NUM>, to be passed back or ejected by a voltage applied by the control transistor <NUM>. This action can be described as "flicking" or "rejecting" and occurs by using a control voltage, such that an analyte passing from one side of the structure <NUM> through the passage <NUM> is modified - either stopped, reversed or accelerated. A control voltage can be applied to each nanopore <NUM>, individually, because each nanopore sensor <NUM> is individually addressable for controlling and sensing. To be clear, the application of a control signal to the sensor electrode <NUM> in each nanopore sensor <NUM> means that the voltage near the nanopore <NUM> at each pixel <NUM> can be controlled.

The control voltage can be applied to alter the movement of an analyte through the nanopore <NUM> in response to at least one condition from conditions including: when a blocked nanopore <NUM> is detected; when the analyte detected is no longer of interest and is to be ejected for the purposes of enabling another sample to be received and measured; and to alter the rate at which an analyte is induced in to or out of the nanopore <NUM>.

An electronic sensor, inevitably, has capacitances, resistances and inductances associated with the path along which the sensor signal travels, which may be referred to as parasitics. These are due to the properties of the materials the sensor is constructed from, the geometry of the sensor, and the methods by which it is feasible to fabricate the sensor. Without any kind of capacitance compensation, these parasitics (most commonly the resistances and capacitances) interact to limit the bandwidth of the signal. In the simplest case, a resistor-capacitor circuit will limit the bandwidth to <NUM> / (<NUM>π R C).

<FIG> is an alternative schematic of <FIG> that illustrates the resistor model <NUM> of the nanopore <NUM> and the channel <NUM> and further includes a compensation circuit <NUM> connected to the voltage divider. According to some embodiments, a compensation circuit <NUM> has an inline amplifier <NUM>, with gain G, connected to the output of the sensor electrode <NUM>, which is influenced by the parasitic input capacitance <NUM>, also known as stray capacitance. The output of the inline amplifier <NUM> has a feedback loop connected to its input, said feedback loop having a feedback amplifier <NUM>, with gain H, and a capacitor C representing compensation capacitance Ccompensation.

A parasitic input capacitance <NUM> is shown arranged in parallel with the resistor representing the channel <NUM>, which represents parasitic capacitance in at least one of: the membrane <NUM> in which the nanopore <NUM> rests; the fluidic walls of the channel <NUM>; the sensor electrode <NUM>; and a trace capacitance associated with a connector or wire-bond. The sensor electrode <NUM> is, in effect, connected to the mid-point in the voltage divider between the nanopore <NUM> and channel <NUM> and connected to the compensation circuit <NUM>. The connection to a reverse or flicking voltage is represented by a flicking switch <NUM>, such as a FET. An optional guard switch <NUM> is shown implemented between the sensor electrode <NUM> and the compensation circuit <NUM>. This guard switch <NUM>, which can be implemented using a FET, can function to isolate the compensation circuit <NUM> and /or any sensing circuitry connected thereto from the flicking voltage applied via the flicking switch <NUM>.

Overall, the compensation circuit <NUM> mitigates the effects of the total parasitic input capacitance <NUM> at the input to the sensing circuit <NUM>. Although the parasitic capacitance resides in various elements of the nanopore sensor <NUM> it can be modelled as shown in <FIG>. Without being bound to a particular theory, a total parasitic input capacitance <NUM> Cp can be considered as a sum of the parasitic capacitances, as follows.

The rate at which the input capacitance charges is proportional to the current flowing through it. In turn, the resistance limits the charging current to a finite value. The compensation circuit <NUM> functions to supply additional current to charge the input capacitance faster, thus increasing the bandwidth.

The compensation circuit <NUM> of <FIG> has an inline amplifier <NUM>, with gain G, connected to the output of the sensor electrode <NUM>, which is influenced by the parasitic input capacitance <NUM>. The output of the inline amplifier <NUM> has a feedback loop to its input, said feedback loop having a feedback amplifier <NUM>, with gain H, and a compensation capacitor C <NUM> representing compensation capacitance Ccompensation.

In some embodiments, the input voltage is amplified and fed back through the compensation capacitor <NUM> so as to provide additional current to charge the total parasitic input capacitance <NUM>. An effective input capacitance of this circuit may be expressed as: <MAT> wherein <MAT>.

The components of the compensation circuit <NUM> are configured such that the total parasitic input capacitance Cp <NUM> is substantially negated or cancelled. In practice, the degree of compensation is limited by dynamic changes in the component values and parameters (e.g. temperature dependence). The compensation circuit <NUM> can compensate for a range of different parasitic input capacitance values if capacitance C, inline gain G or feedback gain H is made adjustable, hence the feedback amplifier <NUM> is illustrated as variable in <FIG>. The gain G can be fixed such that the output from the compensation circuit <NUM> has a consistent gain, therefore either the compensation capacitor C <NUM> and/or the feedback gain H can be varied.

The front-end electronics can reside, at least in part, in the integrated circuit <NUM>, which is figuratively represented in <FIG>. The control circuit <NUM> and/or compensation circuit <NUM> can optionally be incorporated within the integrated circuit <NUM>. The integrated circuit <NUM> or electronic circuit <NUM> is operable to influence the movement of an analyte in the nanopore <NUM>, such as by flicking, by applying a reverse voltage, and amplifying the signal from the nanopore sensor <NUM>. The integrated circuit <NUM> or electronic circuit <NUM> can additionally incorporate further processing of the signal, such as filtering, and may include circuitry to store information locally at the sensor <NUM>, in the case of the integrated circuit <NUM>, for managed communication with an external processor.

Each nanopore sensor <NUM>, such as those shown in <FIG> may be addressable. <FIG> represents the nanopore sensor <NUM> of <FIG> that incorporates the integrated circuit <NUM> within a pixel <NUM> shown in <FIG> and is also addressable via row-selection and column bus. <FIG> is an example of a row-column readout circuit <NUM> connected to each nanopore sensor <NUM> in an array <NUM>, such as the array <NUM> shown in <FIG>, via a row-selection and column bus connection shown in <FIG>. Each nanopore sensor <NUM> is connected to a row decoder <NUM> and column readout <NUM> via an analogue to digital converter (ADC) <NUM>. The row-column readout circuit <NUM> can be connected to the integrated circuit <NUM> of each nanopore sensor <NUM> or group of nanopore sensors <NUM> but may be connected directly to the sensor electrode <NUM> in each nanopore sensor <NUM> within the array <NUM>.

The examples above describe the sensor electrode <NUM> being connected to an integrated circuit <NUM> and having the option of dual functionality when a control voltage is applied (i.e. the sensor electrode <NUM> can be used to sense the change of ion flow when an analyte passes through a nanopore <NUM> and create an electrical potential within the passage <NUM> and a potential difference across the passage <NUM> between the cis electrode 132a and/or trans electrode 132b under the control of the control circuit <NUM>). In this case, the sensor electrode <NUM> is directly connected to a control terminal <NUM>, which is a terminal of the integrated circuit <NUM>, for creating the electrical potential within the passage <NUM>, as shown in <FIG> and described further below.

In some implementations, the sensing and control functions in each nanopore sensor <NUM> may be implemented by separate electrodes, for example as follows. <FIG> shows a sensor electrode <NUM> and control electrode <NUM> arranged like an annulus, while <FIG> are cross-sectional schematics of nanopore sensors <NUM> having configurations in which a control electrode <NUM> is provided in addition to the sensor electrode <NUM>. In this case, the control electrode <NUM> is connected to the control terminal <NUM> of the control circuit <NUM> for creating the electrical potential within the passage <NUM>.

In an example herein, the sensor electrode <NUM> has been described as an annulus, as illustrated in <FIG>. The sensor electrode <NUM> could also be implemented by an exposed wire. The sensor electrode <NUM> could be a nanowire, but can be a larger surface area that occupies, for example, substantially all of the base of a well <NUM>, as shown in <FIG>, or one face of a recess <NUM>. Similarly, a separate control electrode <NUM> could be a nanowire but can have a large surface area, as shown in <FIG>.

From a manufacturability and cost perspective, a basic implementation of a control electrode <NUM> is shown in <FIG>, wherein the annulus footprint - suitable for the base of a well <NUM> - is substantially maintained, while one half of the footprint forms the sensor electrode <NUM> and the other half, which is physically disconnected or decoupled from the sensor side, forms the control electrode <NUM>. There is no wired or solid-state connection between the sensor electrode <NUM> and the control electrode <NUM>. The sensor electrode <NUM> and control electrode <NUM> are shown having two equally sized semi-circle shapes occupying the footprint. The electrodes can be different sizes, and, for example, the control electrode <NUM> can have a greater surface area than the sensor electrode <NUM> to increase the conductivity with the fluid within the passage <NUM>.

Having separate sensor and control electrodes <NUM>, <NUM> can simplify the integrated circuit <NUM> because, by being separate, an extra degree of separation is provided, although they will still be connected through a fluid in the passage <NUM>. However, it can be possible to avoid the need of an isolating switch to protect, for example, the compensation circuit <NUM>, which can form part of the sensing circuit <NUM>, from the voltages applied by the control circuit <NUM>. The sensor and control electrodes <NUM>, <NUM> can be tailored in shape, size and configuration to be optimised for their purpose.

<FIG> indicates how the sensor electrode <NUM> of <FIG> can be divided into a separate sensor electrode <NUM> and control electrode <NUM>. In this example the sensor and control electrodes <NUM>, <NUM> extend in the same plane. In an alternative configuration shown in <FIG> the sensor electrodes <NUM> reside in the cavity <NUM> and extend in a plane extending in parallel with the cis-surface <NUM> and trans-surface <NUM>, while the control electrodes <NUM> extend in the channel <NUM> and extend perpendicularly from said cis- and trans-surfaces <NUM>, <NUM>. In <FIG> the sensor electrode <NUM> is shaped like an annulus while the control electrode <NUM> is shaped like a cylinder. In yet another alternative, as shown in <FIG>, the sensor electrodes <NUM> reside in the cavity <NUM> and extend in a plane extending in parallel with the cis-surface <NUM> and trans-surface <NUM>, while the control electrodes <NUM> extend in the channel <NUM> and in the cavity <NUM>, thus extending in vertical and horizontal planes, as viewed. <FIG>, which is similar to <FIG>, shows the sensor and control electrodes <NUM>, <NUM> formed at the base of the well <NUM>, which can offer easier fabrication.

As described above, an electronic sensor inevitably has capacitances, resistances and inductances associated with the path along which the sensor signal travels, which may be referred to as parasitics, which includes parasitic capacitances. Additionally, or alternatively to the compensation circuit <NUM> described above, the array <NUM> and nanopore sensors <NUM> therein can be fabricated with a guard conductor <NUM> incorporated therein, as shown in <FIG> to <FIG>, while <FIG> shows first and second schematic circuits <NUM> and <NUM> with and without a guard conductor <NUM> in order to illustrate how a guard conductor <NUM> is configured.

In the left-hand, first schematic circuit <NUM> of <FIG>, the parasitic capacitance Cparasitic is shown between two conductive elements <NUM>, <NUM> of the nanopore sensor <NUM>, typically being a conductor, such as the sensor electrode <NUM>, and a conductive substrate of the base layer <NUM>. The first conductive element <NUM> (e.g. sensor electrode <NUM>) may carry a voltage Vsensor and the second conductive element <NUM> (e.g. the conductive substrate) may carry a different voltage Vsubstrate.

Guarding is shown implemented in the right-hand, second schematic circuit <NUM>, wherein guard conductor <NUM> which is a third conductive element is configured between the first conductive element <NUM> carrying the signal and the second conductive element <NUM> such that two parasitic capacitances Cpar1, Cpar2 can be modelled as connected in series. In this second schematic circuit <NUM>, the parasitic capacitance occurs (i) between the first conductive element <NUM> carrying the signal and the guard conductor <NUM>, i.e. Cpar1, and (ii) between the guard conductor <NUM> and the second conductive element204, i.e. Cpar2. A buffer <NUM> (which may be an amplifier) is connected between the first conductive element <NUM> and the guard conductor <NUM> to apply a buffered version of the input signal to the guard conductor <NUM>. As a result, there is no voltage difference across the parasitic capacitance Cpar1 between the first conductive element <NUM> and the guard conductor <NUM>.

For a capacitor, the current is given by <MAT>.

In the right-hand schematic, Vguard = Vsensor, thus <MAT>.

No current flows through the capacitor Cpar1, thus the effective capacitance is zero. The capacitance between the guard conductor <NUM> and the substrate conductors must still be charged, but the buffer <NUM> is able to supply much more current than the high-impedance sensor input, so it charges much faster.

These conditions are met when Vguard accurately follows Vsensor, which depends on the performance of the buffer <NUM> having sufficient bandwidth to enable the capacitance to be nulled. Precise buffers <NUM> with bandwidths of several MHz can be implemented.

<FIG> is analogous to Fig. <NUM>(b) and shows, by way of comparison, a guard conductor <NUM> extending between oxide layers <NUM> along the length of the channel <NUM>, vertically as viewed, and continues horizontally, as viewed, along the top of the base layer <NUM> beneath the sensor electrode <NUM>. Notably, both the sensor electrode <NUM> and the guard conductor <NUM> are connected to the separate electronic circuit <NUM>. In this configuration the guard conductor <NUM> inhibits current flow in the parasitic capacitance between the sensor electrode <NUM> and the substrate of the base layer <NUM>. A conductive guard can include, at least in part, the guard conductor <NUM> and an insulating layer, such as the oxide layer <NUM>, that insulates the guard conductor <NUM> from the conductor being guarded or the conductor being guarded from. The insulating layer is not part of the guard conductor <NUM> and functions to isolate the guard conductor <NUM> from surrounding conductors. The insulating layer, therefore, can be an non-conductive component of the structure <NUM>. The guard conductor <NUM> can be a conductor inserted into the middle of the parasitic capacitances to divide them in two, which is possible because capacitors are by nature insulators, thus the guard conductor <NUM> is located in an existing insulating layer.

The conductive guard, including an insulating layer, can be configured in a number of different configurations, or combination thereof, comprising at least one of: extending over at least a portion of the nanopore layer <NUM> for separating the nanopore layer <NUM> from an analyte in the analyte reservoir <NUM>, as shown in <FIG>, which guards the solution underneath the nanopore <NUM> from the solution above; extending between the at least a portion of the nanopore layer <NUM> and the sense layer <NUM> for separating the sensor electrode <NUM> and integrated circuit <NUM> from the solution in the cis <NUM>, as also shown in <FIG>; extending between the base layer <NUM> and the sense layer <NUM>, at least in part, for separating the sensor electrode <NUM> and integrated circuit <NUM> from the base layer <NUM>, as also shown in <FIG>; and a plurality of conductive guards, as shown in <FIG>, wherein a first conductive guard extends between the walls of the channel <NUM> and the base layer <NUM> and a second conductive guard extends between the sense layer <NUM> and the base layer <NUM>.

In light of the teaching herein a skilled person would appreciate that one of the guard arrangements taught herein, or a combination thereof, could be implemented. It will also be appreciated that the guard conductor <NUM> can be provided in an array <NUM> of nanopore structures, e.g. the array <NUM> of <FIG>.

It is to be noted that guard-based capacitance compensation techniques, shown in <FIG> have the advantage that they generally do not appreciably increase the noise level of the signal. However, such a technique cannot compensate for the membrane capacitance when a potential difference across the membrane <NUM> is used to drive the analyte being studied through the nanopore <NUM>, but it may be possible to drive the analyte by another means, e.g. pressure. A compensation circuit <NUM>, on the other hand, can compensate for the entire input capacitance, but does so at the expense of added noise. The noise gain of the compensation capacitor <NUM> increases with frequency. Therefore, the noise in the input signal is scaled by this feedback gain 'G' and adds to the overall noise. This becomes significant at higher frequencies or when compensating for larger input capacitances. The guard conductors <NUM> shown in <FIG> can, therefore, be implemented in the array <NUM> in any combination and/or in combination with a compensation circuit <NUM>.

The nanopore sensors <NUM> can be manufactured using a number of different techniques and the functions are taught, by way of example with reference to <FIG>, which is indicative of the other sensors taught in the application. Although only one of the nanopore sensors <NUM> of the array <NUM> is shown in <FIG>, the fabrication of an array <NUM> can be understood from the teaching herein. The base layer <NUM> is formed from a standard silicon (Si) wafer that has channels <NUM> formed therein to pass from one side of the base layer <NUM> to the other. Only one channel <NUM> is shown in <FIG>, formed through the Si wafer extending substantially perpendicularly to the surfaces of the wafer. In practice, the array <NUM> has channels <NUM> formed across the wafer using techniques such as photolithography or deep reactive-ion etching (DRIE) or combinations thereof. At least one channel <NUM> is formed for each nanopore sensor <NUM>. Techniques such as thermal oxidation can be used to adjust the diameter of the channel <NUM> to calibrate the aspect ratio, if required. The Si wafer and channels may be embedded in an oxide layer, which may be formed on a silicon wafer, for example.

The example of <FIG> schematically shows a portion of the structure <NUM> having an array <NUM> of nanopore sensors <NUM> (only one of which is shown) and is configured to separate an analyte reservoir <NUM> and outlet chamber <NUM> having drive electrodes <NUM> therein. All of the nanopore sensors <NUM> herein can be located in a structure <NUM> as shown in <FIG>. The nanopore <NUM> lies in the passage <NUM> between the analyte reservoir <NUM> and the outlet chamber <NUM>, which are fluid filled. The passage <NUM> is fluid filled such that the analyte reservoir <NUM> and outlet chamber <NUM> are fluidically connected. To be clear, the nanopore <NUM> lies in a path of fluidic communication between the analyte reservoir <NUM> and outlet chamber <NUM>.

<FIG> show two further examples of a device <NUM> including a structure <NUM>. In each case, the structure <NUM> takes the form shown in either <FIG> or <FIG> including a nanopore layer <NUM>, a sense layer <NUM> and a base layer <NUM>, as described in detail above (although in each case it could be replaced by a structure <NUM> taking the form shown in <FIG>).

In each of the examples of <FIG>, the structure <NUM> separates the analyte reservoir <NUM> and the outlet chamber <NUM> and is connected to a printed circuit board <NUM> but with different configurations as follows.

In the example of <FIG>, the analyte reservoir <NUM> and the outlet chamber <NUM> are each formed by respective gaskets <NUM>, <NUM> which seal against the nanopore layer <NUM> and the base layer <NUM>, respectively. The analyte reservoir <NUM> and the outlet chamber <NUM> may be open as shown in <FIG> or may be closed, for example by respective members extending across the gaskets <NUM>, <NUM>.

In the example of <FIG>, the printed circuit board <NUM> is mounted to the base layer <NUM> by a mechanical bond <NUM> (e.g. adhesive) on the opposite side from the nanopore layer <NUM>. Thus, the printed circuit board <NUM> is disposed outside the outlet chamber <NUM>, as shown in <FIG>. The sense layer <NUM> is connected to the printed circuit board <NUM> by a wire bond <NUM>, or any other suitable electrical connection. The nanopore layer <NUM> has a smaller area than the sense layer <NUM> to provide space for the wire bond <NUM>.

In the example of <FIG>, the printed circuit board <NUM> is mounted to the sense layer <NUM> by a solder bump connection <NUM> (e.g. adhesive) on the same side as the nanopore layer <NUM>. Thus the nanopore layer <NUM> has a smaller area than the sense layer <NUM> to provide space for the solder bump connection <NUM>. The solder bump connection <NUM> provides both mechanical and electrical connection between the printed circuit board <NUM> and the sense layer <NUM>.

In the example of <FIG>, the analyte reservoir <NUM> and the outlet chamber <NUM> are each formed in respective flowcells <NUM>, <NUM> which may be made of any suitable material, for example plastic. The flowcells <NUM>, <NUM> allow flow of fluid into and out of the analyte reservoir <NUM> and the outlet chamber <NUM>.

The flowcell <NUM> that forms the analyte reservoir <NUM> is sealed to the printed circuit board <NUM> around the analyte reservoir <NUM> by a gasket <NUM>, and the printed circuit board <NUM> is sealed to the edges of the nanopore layer <NUM> around the analyte reservoir <NUM> by a sealant <NUM>.

The flowcell <NUM> that forms the outlet chamber <NUM> is sealed to the base layer <NUM> around the outlet chamber <NUM> by a gasket <NUM>.

The examples of <FIG> can be modified in various ways, for example to provide sealing in other locations (e.g. around the outside edge of the base layer <NUM>) and by any suitable means.

The electrical model of a nanopore sensor <NUM> has been described above. More generally, a voltage source, not shown in <FIG>, applies a potential difference between the drive electrodes <NUM> configured in the analyte reservoir <NUM> and outlet chamber <NUM>. The drive electrodes <NUM> impose an electrical potential across the passage <NUM>, including the nanopore <NUM> and channel <NUM>. The nanopore resistance and channel resistance are significantly higher than the overall fluidic resistance of the analyte reservoir <NUM> and outlet chamber <NUM> and, therefore, the nanopore <NUM> and channel <NUM> are the dominant components in an equivalent electrical circuit. As shown in <FIG>, the sensor electrode <NUM> lies between the nanopore <NUM> and channel <NUM> such that it can sense the fluidic electrical potential at the electrical transduction element in the passage <NUM>. In other words, the sensor electrode <NUM> can sense a signal indicative of local electrical potential fluctuations in the passage <NUM>. Although the configuration in <FIG> is an example, the sensor electrode <NUM> can be located in the analyte reservoir <NUM> or outlet chamber <NUM>. The sensor electrode <NUM> can function as the base or gate of a transistor device for measuring electrical potential of the fluid at the location of the sensor electrode <NUM> when a fluid is provided in the passage <NUM>. The sensor electrode <NUM> can detect fluctuations in voltage as a species object, such as a strand of DNA, translocates through the nanopore <NUM>.

The embodiments herein have described a device <NUM> having a single analyte reservoir <NUM> separated from a single outlet chamber <NUM> by the structure <NUM>. In light of the teaching herein alternative arrangements can be implemented and include a device <NUM> having (i) two or more analyte reservoirs <NUM> separated from a common outlet chamber <NUM> by the structure <NUM>, (ii) a common analyte reservoir <NUM> separated from two or more outlet chambers <NUM> by the structure <NUM>, or (iii) two or more analyte reservoirs <NUM> separated from two or more respective outlet chambers <NUM> by the structure <NUM>.

The nanopore layer <NUM> can be formed separately having an array of wells <NUM> that can be formed in a number of ways, one of which is by lithographically patterning a polymer layer. The wells <NUM> in the nanopore layer <NUM> are then aligned with the channels <NUM> of the base layer <NUM> such that each nanopore sensor <NUM> has a passage <NUM> defined by the well <NUM> and channel <NUM>. The well <NUM> shown in <FIG> is substantial in comparison to the nanopore <NUM> located in the membrane <NUM>. The nanopore <NUM> of <FIG> is a biological nanopore in a membrane <NUM> such as an amphiphilic membrane. Alternatively, each nanopore <NUM> can be a solid state nanopore located in a solid-state membrane. Further alternatively, the nanopores <NUM> can be biological nanopores located in a solid-state membrane. In light of the dimensions of the nanopore <NUM> relative to the width of the channel <NUM>, which is greater in diameter, a well <NUM> can be said to form beneath the nanopore <NUM>. The nanopore <NUM>, therefore, defines a part of the passage <NUM> in each of the alternative nanopore configurations.

Any membrane <NUM> may be used in accordance with various aspects described herein. Suitable membranes <NUM> are well-known in the art. The membrane <NUM> can be an amphiphilic layer or a solid-state layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles which form a monolayer are known in the art and include, for example, block copolymers (<NPL>). The copolymer may be a triblock, tetrablock or pentablock copolymer. The membrane <NUM> can be a triblock or diblock copolymer membrane.

Membranes <NUM> formed from block copolymers hold several advantages over biological lipid membranes. Because the triblock copolymer is synthesized, the exact construction can be carefully controlled to provide the correct chain lengths and properties required to form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are not classed as lipid sub-materials; for example, a hydrophobic polymer may be made from siloxane or other non-hydrocarbon-based monomers. The hydrophilic sub-section of block copolymer can also possess low protein binding properties, which allows the creation of a membrane <NUM> that is highly resistant when exposed to raw biological samples. This head group unit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical and environmental stability compared with biological lipid membranes, for example a much higher operational temperature or pH range. The synthetic nature of the block copolymers provides a platform to customize polymer-based membranes for a wide range of applications.

The membrane <NUM> can be one of the membranes disclosed in <CIT> or <CIT>. These documents also disclose suitable polymers.

The amphiphilic molecules may be chemically-modified or functionalized to facilitate coupling of the polynucleotide.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported. The amphiphilic layer may be concave. The amphiphilic layer may be suspended from raised pillars such that the peripheral region of the amphiphilic layer (which is attached to the pillars) is higher than the amphiphilic layer region. This may allow the microparticle to travel, move, slide or roll along the membrane as described above.

The membrane <NUM> may be a lipid bilayer. Suitable lipid bilayers are disclosed in <CIT>, <CIT> and <CIT>.

Methods for forming lipid bilayers are known in the art. Lipid bilayers are commonly formed by the method of <NPL>), in which a lipid monolayer is carried on aqueous solution/air interface past either side of an aperture which is perpendicular to that interface.

Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si<NUM>N<NUM>, Al<NUM>O<NUM>, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses. The solid-state layer may be formed from graphene. Suitable graphene layers are disclosed in <CIT>. <NPL> and <CIT> describe the delivery of proteins to transmembrane pores in solid state layers without the use of microparticles.

Any transmembrane pore may be used. The nanopore <NUM> may be biological or artificial. Suitable nanopores <NUM> include, but are not limited to, protein pores, polynucleotide pores and solid-state pores. The nanopore <NUM> may be a DNA origami pore (<NPL>).

The transmembrane pore can be a transmembrane protein pore. A transmembrane protein pore is a polypeptide or a collection of polypeptides that permits hydrated ions, such as the byproducts of processing a polynucleotide with a polymerase, to flow from one side of a membrane <NUM> to the other side of the membrane <NUM>. In one embodiment, the transmembrane protein pore is capable of forming a nanopore <NUM> that permits hydrated ions driven by an applied potential to flow from one side of the membrane <NUM> to the other. The transmembrane protein pore can permit polynucleotides to flow from one side of the membrane <NUM>, such as a triblock copolymer membrane, to the other. The transmembrane protein pore may allow a polynucleotide, such as DNA or RNA, to be moved through the nanopore <NUM>.

The transmembrane protein pore may be a monomer or an oligomer. The pore can be made up of several repeating subunits, such as at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> subunits. The pore can be a hexameric, heptameric, octameric or nonameric pore. The pore may be a homo-oligomer or a hetero-oligomer.

The transmembrane protein pore typically comprises a barrel or channel through which the ions may flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane β barrel or channel or a transmembrane α-helix bundle or channel. The barrel or channel of the transmembrane protein pore typically comprises amino acids that facilitate interaction with nucleotides, polynucleotides or nucleic acids. These amino acids can be located near a constriction of the barrel or channel. The transmembrane protein pore typically comprises one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate the interaction between the pore and nucleotides, polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention can be derived from β-barrel pores or α-helix bundle pores. The transmembrane pore may be derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1 and hemolytic protein fragaceatoxin C ( FraC). The transmembrane protein pore can be derived from CsgG. Suitable pores derived from CsgG are disclosed in <CIT>. The transmembrane pore may be derived from lysenin. Suitable pores derived from lysenin are disclosed in <CIT>.

The analytes (including, e.g., proteins, peptides, small molecules, polypeptide, polynucleotides) may be present in an analyte. The analyte may be any suitable sample. The analyte may be a biological sample. Any embodiment of the methods described herein may be carried out in vitro on an analyte obtained from or extracted from any organism or microorganism. The organism or microorganism is typically archaean, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista. In some embodiments, the methods of various aspects described herein may be carried out in vitro on an analyte obtained from or extracted from any virus.

The analyte can be a fluid sample. The analyte can comprise a body fluid. The body fluid may be obtained from a human or animal. The human or animal may have, be suspected of having or be at risk of a disease. The analyte may be urine, lymph, saliva, mucus, seminal fluid or amniotic fluid, but can be whole blood, plasma or serum. Typically, the analyte is human in origin, but alternatively it may be from another mammal such as from commercially farmed animals such as horses, cattle, sheep or pigs or may alternatively be pets such as cats or dogs. Alternatively, an analyte can be of plant origin.

The analyte may be a non-biological sample. The non-biological sample can be a fluid sample. An ionic salt such as potassium chloride may be added to the sample to effect ion flow through the nanopore.

The polynucleotide may be single stranded or double stranded. At least a portion of the polynucleotide may be double stranded.

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 polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or other synthetic polymers with nucleotide side chains. The polynucleotide can be any length.

Any number of polynucleotides can be investigated. For instance, the method may concern characterising <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more polynucleotides. If two or more polynucleotides are characterised, they may be different polynucleotides or two instances of the same polynucleotide.

The polynucleotide can be naturally occurring or artificial.

The method may involve measuring two, three, four or five or more characteristics of a polynucleotide. The one or more characteristics can be selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified.

For (iii), the sequence of the polynucleotide can be determined as described previously. Suitable sequencing methods, particularly those using electrical measurements, are described in <NPL>, <NPL>, and <CIT>.

The secondary structure may be measured in a variety of ways. For instance, if the method involves an electrical measurement, the secondary structure may be measured using a change in dwell time or a change in ion current flowing through the pore. This allows regions of single-stranded and double-stranded polynucleotide to be distinguished.

The presence or absence of any modification may be measured. The method can comprises determining whether or not the polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers. Specific modifications will result in specific interactions with the pore which can be measured using the methods described below.

In some embodiments of various aspects described herein, the method may involve further characterizing the target polynucleotide. As the target polynucleotide is contacted with the pore, one or more measurements which are indicative of one or more characteristics of the target polynucleotide are taken as the polynucleotide moves with respect to the pore.

The method may involve determining whether or not the polynucleotide is modified. The presence or absence of any modification may be measured. The method can comprises determining whether or not the polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers.

Claim 1:
A device for nanopore sensing, said device comprising:
a structure (<NUM>) arranged to separate an analyte reservoir (<NUM>) and an outlet chamber (<NUM>), the structure (<NUM>) comprising:
a nanopore layer (<NUM>) incorporating an array of wells (<NUM>), each well (<NUM>) having a membrane (<NUM>) that spans the well (<NUM>) and has a nanopore (<NUM>) provided therein, and each well (<NUM>) having a well outlet (142a); and
a base layer (<NUM>) incorporating an array of channels (<NUM>),
the nanopore layer (<NUM>) and the base layer (<NUM>) being sandwiched together such that respective wells (<NUM>) and channels (<NUM>) are aligned to define an array of nanopore structures (<NUM>), wherein each nanopore structure (<NUM>) comprises a passage (<NUM>) defined at least in part by: a nanopore (<NUM>) and a well (<NUM>) of the nanopore layer (<NUM>), at one side of the passage (<NUM>); and a channel (<NUM>) of the base layer (<NUM>), at the other side of the passage (<NUM>), wherein the well (<NUM>) is fluidly connected to the channel (<NUM>) via the well outlet (142a), and the passages (<NUM>) provide fluid connection through the structure (<NUM>) between the analyte reservoir (<NUM>) and outlet chamber (<NUM>);
drive electrodes (<NUM>) connected respectively in the analyte reservoir (<NUM>) and the outlet chamber (<NUM>) for imposing an electrical potential difference across the passages (<NUM>);
electrical transduction elements (<NUM>), each electrical transduction element (<NUM>) being exposed to the passage (<NUM>) of a respective nanopore structure (<NUM>) for measuring the fluidic electrical potential at that electrical transduction element (<NUM>) in that nanopore structure (<NUM>);
characterised by
control terminals (<NUM>), wherein either:
each control terminal (<NUM>) is connected to the electrical transduction element (<NUM>) of a respective nanopore structure (<NUM>); or
the structure further comprises control electrodes (<NUM>), each control electrode (<NUM>) being exposed to the passage (<NUM>) of a respective nanopore structure (<NUM>), and each control terminal (<NUM>) is connected to the control electrode (<NUM>) of a respective nanopore structure (<NUM>), and
the device is configured so that application of a control signal to a control terminal (<NUM>) alters the electrical potential difference across the respective nanopore structure (<NUM>) between the control terminal (<NUM>) and at least one of the drive electrodes (<NUM>).