Nanopore sensing device and method of operation and method of fabrication thereof

Abstract

To provide a nano hole detection device.SOLUTION: A nano hole detection device includes a structure (100) arranged so as to separate an analyte reservoir (106) from an exit chamber (108). The structure (100) includes the array of nano hole structures (104); each nano hole structure (104) includes a passage (114) for connecting a fluid passing a structure between the analyte reservoir (106) and the exit chamber (108); a control terminal (156) is arranged so as to apply a control signal to change a potential difference over the nano hole structure (104); and an electronic circuit (152) is constituted so as to detect a signal from an electric conversion element (126) in each nano hole structure (104).SELECTED DRAWING: Figure 3(a)

Description

[Technical Field]

[0001] The present invention relates generally to devices for nanopore sensing having arrays of nanopore structures that can be configured as nanopore sensors, and methods for operating nanopore sensors or for fabricating arrays of nanopore structures. [Background technology]

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

[0003] An alternative to biological nanopore devices such as the MinION™ is a solid-state nanopore device. Figure 1 shows a portion of a single sensor device 2 having a solid-state nanopore 4 as disclosed in WO 2016 / 127007, the entire contents of which are incorporated herein by reference, in which an analyte 6 passes from a cis reservoir 10 through a body 8 and into a fluid passageway 12 through the solid-state nanopore 4. A signal is read via a sensor 16 located near the solid-state nanopore 4. Electrodes 18 are provided in the cis reservoir 10 and the trans reservoir 14 to guide the analyte 6 through the solid-state nanopore 4.

[0004] The performance of solid-state nanopore sensors is limited by their tolerances, which can arise as a result of variations in the sensing components, fabrication techniques, and formation of the nanopore or sensor assembly. These and other factors impair the bandwidth, sensitivity, and ability to control such nanopore sensors.

[0005] In some aspects, the present disclosure is directed to overcoming problems associated with implementing nanopore sensor arrays having multiple nanopore sensors. Summary of the Invention [Problem to be solved by the invention]

[0006] The inventors have sought to improve upon known nanopore sensing devices by providing, in some embodiments, the ability to control analyte movement while simultaneously improving measurement accuracy by mitigating factors that interfere with the measurement, such as noise caused by parasitics and contamination of the sensing components. Furthermore, the improved devices allow the nanopore structures, and nanopore sensors implemented from the nanopore structures, to be efficiently formed into large arrays without interfering with the control or performance of the array. [Means for solving the problem]

[0007] In a first aspect, the present invention provides a device for nanopore sensing, said device comprising: a structure arranged to separate an analyte reservoir and an exit chamber, the structure comprising an array of nanopore structures, each nanopore structure comprising a passage for fluid connection through the structure between the analyte reservoir and the exit chamber; a driving electrode connected within the analyte reservoir and the exit chamber, respectively, for imposing a potential difference across the passageway; electrical transducer elements, each connected or exposed to a passage of a respective nanopore structure for measuring fluid potential at the electrical transducer element within the nanopore structure; and control terminals, each control terminal connected to a respective nanopore structure for applying a control signal to vary the potential difference across the nanopore structure.

[0008] The structure can be a support structure. The nanopore structures can be disposed within and / or on a corresponding array of passages. Each nanopore structure can have an opening that forms a portion of the passage. Each nanopore structure in the array of nanopore structures has a respective passage. A control terminal can be connected to each passage in the structure to apply a control signal to vary a fluid potential difference distribution across the respective nanopore structure. When fluid is supplied such that there is fluid connection between the driving electrode and the nanopore structure, a control signal applied to the nanopore structure can vary the potential difference across the nanopore structure relative to the driving electrode. The control terminal can be connected to an electrical transduction element. The control terminal can be switchably connected to the electrical transduction element.

[0009] The nanopore structure of the array may have a nanopore, may be capable of supporting a nanopore, or may be capable of supporting a membrane having a nanopore.

[0010] When operated as a nanopore sensing device, the device comprises an array of nanopores.

[0011] When a fluid is applied, the fluid potential can be measured at the electrical transducer element, and when a fluid is applied, the fluid electrical distribution across the nanopore structure can be altered.

[0012] In operation, fluids are present in the analyte reservoir, the outlet chamber, and the passageways of the device where the reservoir and chamber are fluidly connected. The fluids in the reservoir, chamber, and passageways of the nanopore structure can be different fluids.

[0013] The nanopore structure can comprise an opening having a width on the nanometer dimension, which can be a through-hole in a solid support, such as a solid-state nanopore.

[0014] Alternatively, in one embodiment, the nanopore structure may be a structure capable of supporting a nanopore to provide a passageway of nanometer dimensions. In this embodiment, the nanopore structure may have an opening of micrometer or nanopore dimensions. Exemplary nanopore structures that can be used to support a nanopore are disclosed in WO2014 / 064443, the entire contents of which are incorporated herein by reference. An example of a nanopore that can be supported by the nanopore structure is a biological nanopore, such as a protein nanopore. The nanopore can be provided in a membrane, such as an amphiphilic membrane. The membrane can be supported by the nanopore structure.

[0015] When used for nanopore sensing, the device can comprise an array of nanopores.

[0016] The analyte reservoir can function to receive an analyte for detection by the nanopore array. The exit chamber can function to receive an analyte that passes through the nanopore array.

[0017] The nanopore, if present, separates the cis and trans sides of the device: the analyte reservoir can be considered the cis side of the device, and the analyte exit chamber can be considered part of the trans side.

[0018] The device can be provided with or without fluids. The fluids in the analyte reservoir, the exit chamber, and the passage of the nanopore structure can be different fluids.

[0019] In a further aspect, the invention provides a structure comprising an array of nanopore structures, each nanopore structure comprising a passage for fluid connection through the structure. Each nanopore structure has an electrical transduction element, each element connected to or exposed to a passage of the respective nanopore structure for measuring a fluid potential at the electrical transduction element within the nanopore structure. Each nanopore structure also has a control terminal, each control terminal connected to the respective nanopore structure for applying a control signal to vary the fluid potential difference distribution within the passage or across the respective nanopore structure.

[0020] The structure can be a support structure. The nanopore structures can be disposed within and / or on a corresponding array of passages. Each nanopore structure can have an opening that forms a portion of the passage. Each nanopore structure in the array of nanopore structures has a respective passage. A control terminal can be connected to each passage in the structure for applying a control signal to vary the fluid potential difference distribution across the respective nanopore structure. Each opening in the array can be associated with a respective electrical transduction element and control terminal.

[0021] Each nanopore structure in the array of nanopore structures can be considered a pixel, with each pixel comprising an aperture, an electrical conversion element, and a control terminal. The array of pixels can be arranged as a linear grid, similar to the arrangement of pixels on a television screen. When present in a nanopore structure, the nanopore forms a portion of a passageway, i.e., one section of the passageway is nanometer wide. The nanopore can be a solid-state nanopore, i.e., a nanopore-width opening is provided in a solid support. Alternatively, the nanopore can be a hybrid nanopore, in which a biological nanopore is provided within an opening in a solid support. The biological nanopore can be supported within an amphiphilic membrane. The amphiphilic membrane can be supported by pillars, as disclosed in WO 2014 / 064443. Nanopore structures capable of supporting nanopores can have openings with widths wider than the nanopore dimensions, such as micrometer dimensions. The nanopore structure can comprise a means for supporting the amphiphilic membrane. The analyte reservoir can be used to store an analyte, such as an analyte, for analysis. The analyte can pass through a nanopore in the nanopore sensor of the array. After passing through the nanopore, the analyte can remain in the passageway or exit the passageway into an exit chamber. When fluid is supplied to the analyte reservoir, the exit chamber, and the passageway of the array of nanopore structures, the driving electrode can impose a potential difference across the passageway. The driving electrode can provide a potential difference across the opening to induce the charged analyte to pass through the nanopore of the array. The potential difference can be changed to change the speed or direction of movement of the analyte.

[0022] Each electrical transducer element in the array functions as a sensor electrode. Changes in ionic current flow through the nanopore cause variations in electrical potential caused by the changes in ionic current flow, which can be measured to determine the presence or properties of an analyte. The fluid within the device, which can be aqueous, can contain ions. Multiple analytes can be transferred.

[0023] The driving electrodes serve to provide a common potential difference across the array of nanopores, allowing multiple analytes to be measured simultaneously within the array, with measurements taking place at electrical transduction elements within each nanopore structure.

[0024] In some embodiments, each nanopore structure can have an associated control terminal, which can be an independent connection from the structure to an externally generated control signal, allowing a potential to be applied independently of changes in the potential difference across other nanopore structures in the array.

[0025] The control signal can be generated within the nanopore structure in response to an external trigger or switch. Alternatively, the control signal can be generated from circuitry internal to the nanopore structure. The control signal has the effect of changing the voltage level of each nanopore structure. The control signal can be applied via an electrical transduction element to modify the voltage between the passage and the drive electrode. Additionally or alternatively, the control signal can be applied via an electrical connection, such as a control terminal or an additional control electrode within the passage.

[0026] The device can have a single drive electrode in electrical communication with the analyte reservoir and a single drive electrode in electrical communication with the exit chamber, the drive electrodes serving to provide a common potential difference across the nanopore array.

[0027] Alternatively, the device may include multiple drive electrodes on the cis and / or trans sides of the device.

[0028] Application of control signals to individual nanopore structures can function to change the potential difference across the nanopore structure and between the nanopore structure and the driving electrode. By way of example, the driving electrode in the analyte reservoir can have a voltage level of -0.1 volts, while the driving electrode in the exit chamber can have a voltage level of 0.2 volts, resulting in a potential difference of 0.3 volts across the passages of the array. Application of control signals to impose a voltage of -2 volts on the nanopore structure results in a potential difference of -1.9 volts and -1.8 volts between the nanopore structure and the cis and trans electrodes, or respectively.

[0029] The electrical transducer element of each nanopore can be directly connected to the control terminal. In doing so, the electrical transducer element can function as both a sensor electrode and a control electrode. This can be accomplished by providing the electrical transducer element with two terminals, one for connecting to the sensing circuitry and the other for connecting to the control circuitry. In fact, the sensing circuitry and the control circuitry can be within the same circuit or component. Any circuitry can be located outside the structure and connected to the structure via wire bonds, for example.

[0030] The control terminals can be configured to apply a control signal to vary the potential difference from the drive electrodes to each respective nanopore structure in response to measurement of the fluid potential at the nanopore structure by the electrical transduction element, and application of the control signal can be configured to vary the potential difference between at least one of the control terminals and at least one of the drive electrodes.

[0031] A control signal applied to a control terminal of a nanopore structure can change the magnitude and / or polarity of the potential difference between the nanopore and the driving electrode, thereby changing the rate at which an analyte passes through the nanopore structure or changing the direction of movement of the analyte.

[0032] A control signal may be connectable to a plurality of nanopore structures to simultaneously vary the potential difference between the connected control terminal and at least one of the drive electrodes.

[0033] The control signal can be applied for purposes other than rejecting the analyte or controlling the rate and / or direction of its movement. For example, the control signal can be applied to induce insertion of a biological nanopore into a membrane supported by the nanopore structure. The electrical transduction element can be connected to a measurement circuit to read the signal received from the electrical transduction element. The nanopore structure can be provided with a switchable connection to the measurement circuit. The switchable connection can disconnect the measurement circuit before applying the control signal. In this way, the control signal can be disconnected from the measurement circuit, preventing the control signal from affecting the performance of the measurement circuit.

[0034] In other words, the electrical transduction elements may be isolable prior to application of the control signal. Individual electrical transduction elements in each nanopore structure may be selectively isolated prior to application of the control signal.

[0035] The control signal can be applied for a variety of purposes.

[0036] The control signal can be applied independent of the measurement of an analyte, for example, a control signal can be applied to a membrane supported by a nanopore structure to induce the insertion of a biological nanopore into the membrane.

[0037] A control signal can be applied to the nanopore structure in response to a measurement by the electrical transduction element.

[0038] For example, a control signal can be applied to unblock the nanopore when the device determines that passage through the nanopore is blocked, e.g., by an analyte. A control signal can then be applied to unblock the passage.

[0039] The device can determine whether a nanopore is blocked by measuring the change in potential caused by the inhibition of current flow through the nanopore. In the absence of interaction between the analyte and the nanopore, the ionic current flow through the nanopore due to the presence of ionic salts in the aqueous sample may be referred to as the open pore current. When an analyte interacts with the nanopore, the ionic current flow through the pore decreases, and the variation in the decrease in ionic current can be measured as the change in the potential of the sensor electrode over time as the analyte, such as DNA, moves through the nanopore. For example, blockage of the nanopore due to immobilization of the analyte within the pore causes a decrease in ionic current flow, the value of which changes little over time. In a further example, a control signal can be applied to exclude analytes that are not or no longer of interest from the nanopore. Measurements can be performed in real time, allowing a decision to exclude an analyte, such as a polynucleotide, before it has been fully measured.

[0040] For the aforementioned devices for sequencing polynucleotides, such as the MinION™ device, the current through the nanopore is measured under an applied potential difference between a respective array of electrodes on one side of each nanopore and a common electrode on the other side of the analyte reservoir nanopore. Because each nanopore has an associated electrode, the potential difference across each nanopore in the array can be individually controlled to eliminate analytes. In the embodiments described below, there are various advantages associated with performing measurements of the local potential at each nanopore with an electrical transducer element. The driving electrodes serve to provide the potential difference across the nanopore array, not to measure the analyte. As a result, individual control of the potential difference at the nanopores by the driving electrodes is not possible. However, it is possible to individually control the potential difference across each nanopore using a control terminal.

[0041] The array of nanopore structures can have circuits, each circuit associated with a respective nanopore structure and connected to an electrical transduction element. Each circuit can be configured to modify and / or process signals received from the electrical transduction elements. The circuit can also apply control signals to the electrical transduction elements. The circuit can isolate the control signals applied to the electrical transduction elements from other sensing and processing functions.

[0042] Each circuit can reside within a pixel of the nanopore structure. Each circuit can be addressable. Each nanopore structure can be addressable. The addressing capability can allow an external processor to communicate with the nanopore structure to receive measurement information and / or control the movement of analytes within the passageways. In this manner, measurement and control of sensing in each individual passageway can be independently controlled. The circuitry can be provided on or embedded within the support structure.

[0043] Each electronic circuit can be associated with a group of nanopore structures. For example, an electronic circuit can be shared by a group of four nanopore structures. Sensing and control of the nanopore structures within a group can be multiplexed. In this way, the electronic circuit can be addressable, and multiplexing can be used to control individual nanopore structures.

[0044] Each circuit can be associated with a respective nanopore structure or group of nanopore structures. Each circuit can be connected to a control terminal and / or an electrical transduction element such that the circuit is configured to vary the potential imposed by the drive electrodes at the respective nanopore structure in response to measurements at the electrical transduction element and / or measurements from an external processor attached thereto.

[0045] The structure can have a nanopore layer incorporating nanopores and / or wells for supporting a solid film or membrane having nanopores. When nanopores are provided, the nanopore structure can operate as a nanopore sensor. The nanopore layer can be provided with nanopores after the nanopore structure is created. The nanopores can be provided by the user after a device having the nanopore structure is provided to them. The nanopore layer can be replaced, thereby making the device recyclable. The nanopore structure can also include a base layer incorporating channels. The nanopore layer and base layer can be sandwiched or laminated together so that the nanopores and / or wells align to define a passageway. At least one of the electrical transduction element, circuitry, or control terminal is disposed on or between the outer surface of the structure. An individual nanopore structure can be composed of a single structure or one or more substructures connected to each other. The single structure or substructure can be planar or sheet-like.

[0046] Each nanopore structure can be defined by its passageway. The passageway can fluidly connect the cis and trans. The passageway can be formed by a formation within each nanopore structure, for example, formed by a nanopore layer for supporting the nanopore, a layer having through-holes, and a base layer having channels that function as through-holes. The through-holes in the nanopore layer and base layer are aligned to form the passageway.

[0047] The electrical transduction element defines a portion of the passageway. By way of example, the electrical transduction element may be sandwiched or laminated between the nanopore layer and the base layer, although it may be located elsewhere in the passageway. It may be configured around the passageway provided there is a fluid connection, which may be a direct fluid connection between the electrical transduction element and a nanopore disposed in the nanopore layer.

[0048] The electrical transduction element and / or circuitry can be implemented on the sense layer. The sense layer can be a substructure. The sense layer can be sandwiched or embedded between a nanopore layer and a base layer, the sense layer having through-holes that align with the through-holes in the nanopore layer and base layer. For clarity, the nanopore layer, sense layer, and base layer can be substructures that are stacked to provide an array of nanopore structures.

[0049] When the nanopore is disposed within the nanopore structure, it forms a portion of a passageway. Rejection of the analyte can be managed using a control signal that functions to control the movement of the analyte within the nanopore, e.g., to reject the analyte from the nanopore. A nanopore within the passageway can become blocked. Blockage of the nanopore can be detected, and a control signal can be applied to the nanopore structure to remove the blockage.

[0050] The nanopore can be a solid-state nanopore, i.e., a nanometer-wide pore, located within a solid membrane. The membrane can be a nanopore layer or a membrane disposed on a nanopore layer. The solid-state nanopore can be located on a nanopore layer. Alternatively, the nanopore can be a biological nanopore located in a solid film or membrane. Alternatively, the nanopore can be formed using a well into which a membrane, such as an amphiphilic membrane or lipid bilayer, can be formed so that the nanopore can be inserted into the membrane. In each of these nanopore examples, one nanopore can be provided for each nanopore structure in the array.

[0051] The inventors have also attempted to improve the architecture of nanopore sensors, particularly where such improvements can optimize sensitivity and performance. The inventors generally attempt to achieve this by providing a structure having nanopore structures, with nanopore structures disposed within the structure providing fluid communication from one side of the structure to the other via a passageway in each nanopore structure. In this manner, the structure can separate cis and trans. Each nanopore structure has a sensor electrode. To minimize attenuation of the signal derived from the sensor electrode and avoid any adverse effects of noise on that signal, each nanopore structure is provided with circuitry for processing the signal from the sensor electrode before communicating the processed signal for further processing and / or analysis. The circuitry can be embedded in the nanopore structure. The circuitry can occupy the same footprint as the nanopore structure, such that the nanopore structure can be considered an active pixel. Nanopore structures with their own circuitry complement the improved control mechanisms disclosed herein by generating and applying control signals locally, thus minimizing the impact of the control signals on other nanopore structures in the array.

[0052] Thus, in a second aspect, the present invention provides a device having nanopore structures for sensing an analyte, the nanopore structures being configured within a structure arranged to separate an analyte reservoir and an outlet chamber, each nanopore structure providing a passage for fluid connection through the structure between the analyte reservoir and the outlet chamber, each nanopore structure comprising an electrical conversion element and electronic circuitry configured to detect and optionally amplify a signal from the electrical conversion element, and each of the structures configured to one or more of store, transmit, process and communicate at least a portion of the signal to a connectable processor.

[0053] The nanopore structures may be included as part of an overall structure in which individual nanopore structures are joined together.

[0054] The structure can be configured to separate an analyte chamber for receiving an analyte and an exit chamber for collecting the analyte. Actuation electrodes can be connected within the analyte reservoir and the exit chamber, respectively, to impose a potential difference across a passage within the nanopore structure. If a nanopore is provided, the nanopore structure can function as a nanopore sensor, and the device can be a nanopore sensing device.

[0055] Each nanopore structure in the array can further comprise a compensation circuit. The compensation circuit function can be integrated with other processing functions of the circuitry within the nanopore structure. The compensation circuit can have a variable gain amplifier and / or a variable capacitor in its feedback loop.

[0056] As described in the first embodiment, the structure may have control terminals for applying a control signal to vary the potential difference across the nanopore structure. A control signal may be switchably applied to the control terminals to adjust a configurable voltage level imposed on the pore.

[0057] Nanopore structures incorporating circuitry, which may include compensation circuitry, can be packaged in a defined footprint or pixel space. An array of pixel-spaced nanopore structures can be arranged in a mosaic-like array.

[0058] The signal from the electrical transduction element can be processed, at least in part, within the nanopore structure itself, thereby allowing the signal to be processed or managed locally. For example, the signal can be amplified locally to minimize attenuation or noise affecting the signal before analysis elsewhere. The circuitry can also store the signal, the signal value, or data derived from the signal. In this manner, information derived from the nanopore structure can be communicated to a processor remote from the nanopore structure upon request. Each nanopore structure, or the circuitry within the nanopore structure, can be addressable. The circuitry can be connected to an analog-to-digital converter (ADC) located outside the nanopore structure.

[0059] The inventors have further sought to provide structures that generally improve the manufacturability of arrays of nanopore structures, while improving sensitivity and performance. The arrays of nanopore structures herein can not only provide improved nanopore structures, but also complement the integration of control functions and local control.

[0060] Thus, in a third aspect, the present invention provides a device having an array of nanopore structures, the structures may be configured into a sheet comprising a nanopore layer having an array of nanopores and / or an array of wells for supporting the nanopores, and a base layer having an array of channels, the base layer being sandwiched or laminated to the nanopore layer to form the sheet, the nanopores and / or wells being aligned with the channels, and each of the nanopore structures comprising a passageway, each passageway being defined at least in part by one of the nanopores and / or one of the wells in the nanopore layer on one side of the passageway, the channel in the base layer on the other side of the passageway, and an electrical transduction element.

[0061] In another aspect, the present invention provides an array of nanopore structures, each of which, when provided with a nanopore, functions as a nanopore sensor, each of which has a through-hole defined by a nanopore, or well, channel, and electrical transduction element, if provided.

[0062] The sheet can be a substantially planar array of nanopore structures. When the nanopore structures are provided with nanopores, they can function as nanopore sensors. The sheet can be configured in the device to separate an analyte reservoir and an outlet chamber. The analyte reservoir and the outlet chamber can contain a fluid. The passage can be filled with a fluid to provide a fluid connection between the analyte reservoir and the outlet chamber.

[0063] Constructing the nanopore layer and base layer as separate layers can improve the scalability of the sheet. These layers can facilitate device assembly and therefore reduce manufacturing costs. Layering the sheet can organize the components of the nanopore structure in an efficient manner. Furthermore, having different components of the nanopore structure on different layers can enable optimization of the formation or configuration of those components. Often, the process used to fabricate one component is incompatible with or adversely affects the fabrication of another component. Furthermore, the optimal material for forming one component may differ from the optimal material for forming another component. For example, the array of nanopores and / or the array of nanopore wells can be formed separately from the base layer. The nanopore layer and base layer can comprise different materials. Separate layers can enable optimal configuration and / or placement of the components of the nanopore structure.

[0064] The provision of a layer may allow the layer to be replaceable. The nanopore layer may be removably attached. In this way, for example, if the nanopore layer becomes contaminated, the nanopore layer may be replaced with a replacement nanopore so that the device can be recycled.

[0065] Each nanopore structure in the sheet is defined by a passageway. The various components of the nanopore structure, namely, the nanopore or nanopore well, the electrical transduction element, and the channel, form the passageway. The nanopore layer need not have nanopores, but can have nanopores. The nanopores can be configured on wells in the nanopore layer, and in doing so, this additional nanopore on the well also forms an element of the passageway.

[0066] An electrical transduction element in each passage can be disposed between the nanopore layer and at least a portion of the channel, the electrical transduction element having a nanopore structure and configured with a connection for measuring the electrical potential of the fluid at the location of the electrical transduction element when the fluid is supplied into the passage.

[0067] The electrical transducer element can exhibit a characteristic indicative of the fluid potential at the electrical transducer element in the passageway connecting the cis and trans reservoirs via the fluid in the passageway. The electrical transducer element can be an electrical connection. It can be located within the cis or trans reservoir, on the surface of the nanopore structure, at a location within the passageway, or at another location within the nanopore structure.

[0068] The electrical transduction element can be a device or region of a device and / or a combination of circuitry, wires, or circuit elements that senses the fluid potential at the electrical transduction element of the device. Additionally or alternatively, a circuit can be provided as the transduction element to generate a signal indicative of the local electrical potential.

[0069] As described, the device can have an analyte reservoir and an outlet chamber at least partially separated by a sheet. The analyte reservoir functions as a sensor and can hold an analyte to be analyzed by the nanopore structure, if a sensor is provided. A passage in the nanopore structure of the array connects the analyte reservoir to the outlet chamber. The interface between the analyte reservoir and the outlet chamber can be a passage, or more specifically, a nanopore in the nanopore sensor, i.e., a nanopore structure with a nanopore.

[0070] The device can have actuation electrodes connected to the analyte reservoirs and the exit chambers for imposing a potential difference across the array of passages between the analyte reservoirs and the exit chambers.

[0071] The sheet can be substantially planar. The surface of the sheet, the structure incorporating the array of nanopore structures, has a cis-surface on the nanopore layer facing the analyte reservoir and defining a cis-face, and a trans-surface on the base layer facing the exit chamber and defining a trans-face. The array of electrical transduction elements can be at least partially embedded within the sheet between the cis- and trans-faces. The electrical transduction elements of the array can be sandwiched between the nanopore layer and the base layer.

[0072] Each nanopore structure in the array can have a well formed at a first end of the passageway. The nanopore can be configured at the first end of each well. An electrical transduction element can be configured on the opposite side of the well from the nanopore. The well can be larger in size than the nanopore, allowing for an increased volume of fluid surrounding the nanopore. For clarity, the diameter of the well can be larger than the diameter of the nanopore. The nanopore can reside within a membrane spanning the well. The membrane can be a solid membrane, an amphiphilic membrane, or a lipid bilayer. The nanopore defines a portion of the passageway. Access to and egress from the well is via the nanopore and the well outlet.

[0073] The wells can be configured to support a fluid membrane, such as a polymer membrane or a lipid bilayer. The nanopore layer can be made of a different material than the base layer. By using a different material for the nanopore layer, the material can be selected to have a surface energy that optimizes the formation of a membrane across the well to support the nanopore.

[0074] The electrical transduction element can be a sensor electrode that can be directly connectable to the base or gate of a transistor device for measuring variations in the electrical potential of the fluid at the location of the electrical transduction element when the fluid is supplied into the passageway. As described herein, a nanopore structure with a nanopore forming a portion of the passageway functions as a nanopore sensor, and sensing is performed by the electrical transduction element.

[0075] The electrical transduction elements of the nanopore structures of the array can be connected to edge connectors or wire bonds. The connectors can provide connections to measurement circuitry off the sheet, i.e., connections separate from the array of nanopore structures. The connectors can be connected to vias that lead to connections at the edge of the sheet for subsequent connection to measurement circuitry off the sheet. The transistor devices can be field effect transistors.

[0076] Thus far, we have described a sheet having a nanopore layer and a base layer. The electrical transduction elements can be layers within the sheet or can have elements sandwiched between the layers. However, the device sheet can further include a sense layer having an array of electrical transduction elements, with the sense layer sandwiched between the nanopore layer and the base layer. The electrical transduction elements can be formed on the sense layer. The electrical transduction elements can have exposed portions for connecting to fluid in the passages and embedded portions embedded within the sheet. Additionally or alternatively, the electrical transduction elements can have connection portions for connecting to measurement circuitry separate from the sheet. Incorporating the electrical transduction elements into or on the sense layer allows for the formation of the electrical transduction elements to be separated from the fabrication of the other layers. The sense layer can be fabricated using different materials, processes, and / or techniques than the other layers.

[0077] The electrical transduction element can at least partially cover the wall of the passageway. The electrical transduction element can, in cross section, cover a portion of the wall of the channel. The electrical transduction element can form an annulus around the base of the passageway and / or a well or cavity within the passageway.

[0078] The electrical transduction element can be formed on one surface of the sense layer. The sense layer can be sandwiched between a base layer and a nanopore layer, with the electrical transduction element aligned with the nanopores or wells of the nanopore layer and the channels of the base layer. When aligned, the surface of the sense layer can expose the electrical transduction element to the nanopore layer, such that the nanopore layer is formed or disposed on the surface having the electrical transduction element, and in this arrangement, the electrical transduction element can be said to face the nanopore layer. Alternatively, when aligned, the surface of the sense layer can expose the electrical transduction element to the base layer, such that the electrical transduction element is formed or disposed on the surface of the base layer, and in this arrangement, the electrical transduction element can be said to face the base layer.

[0079] The electrical transduction element can at least partially form a surface of the sense layer around the passageway and can have an exposed portion disposed facing the outlet chamber. The exposed portion can form a portion of a wall of a cavity formed in the sense layer between the well and the channel. The cavity can allow a larger area of ​​the sensor electrode to be exposed to fluid in the passageway, which can improve the sensitivity of the sensor electrode.

[0080] The electrical transduction element can have an opening that forms a portion of the passageway and the exposed portion, and in cross section, the ratio of the size of the exposed portion of the electrical transduction element to the size of the opening is 1:1. The ratio can be about 5:1.

[0081] The electrical transduction element may have an opening that forms a portion of the passageway and the exposed portion, and the ratio of the size of the exposed portion of the electrical transduction element to the size of the opening in a plan view is 1:1. The electrical transduction element may have an opening that forms a portion of the passageway and the exposed portion, and the ratio is about 5:1. The opening may be circular.

[0082] The electrical transduction element can have a large exposed area to increase its exposure to fluid within the passageway and increase the element's sensitivity to voltage fluctuations caused by analytes passing across or through the nanopore within the passageway.

[0083] The sense layer can incorporate electronic circuitry for each nanopore structure. The circuitry can be connected to the electrical transduction elements to modify and / or process the signals received from the electrical transduction elements. Incorporating electronic circuitry within each nanopore structure can then locally process the signals from the electrical transduction elements to reduce information attenuation and / or any adverse effects of noise on the resulting signals. Each electrical circuit within each nanopore structure can process signals from the sensor electrodes before the processed signals are communicated off-sheet for further processing and / or analysis. Incorporating circuitry into the sense layer can embed the circuitry within the nanopore structure. The circuitry can occupy the same footprint as the nanopore structure, such that the nanopore structure can be considered an active pixel. Nanopore structures with their own circuitry complement the improved control mechanisms disclosed herein by generating and applying control signals locally, thus minimizing the impact of the control signals on other nanopore structures in the array. The circuitry within the sense layer of the nanopore circuit can be a compensation circuit.

[0084] The electronic circuitry can be configured to detect a change in resistance of the nanopore at each passage when an analyte passes through or is adjacent to the nanopore, and the circuitry can detect a change in resistance detected via a fluid within the sensor.

[0085] Although the device has been described as being suitable for sensing an analyte, it should be understood that the analyte is one that can be measured using the nanopore. By way of example, the analyte may be a protein, a polymer, a polynucleotide, etc.

[0086] The electronic circuitry can detect a change in electrical potential at the electrical transducer element as the polymer passes through the nanopore, convert the change in electrical potential into a voltage signal, and amplify the voltage signal. The electronic circuitry can filter the signal. The electronic circuitry can sample and / or digitize the signal obtained from the electrical transducer element.

[0087] Each nanopore structure can have a plurality of electrical transducer elements corresponding to each respective nanopore structure. Similarly, each nanopore structure can have a plurality of circuits corresponding to each respective nanopore structure and / or electrical transducer elements provided within the nanopore structure. Each of the electrical transducer elements and / or circuits can be configured in an addressable array. Each nanopore structure can have two or more sensor electrodes. Two or more sensor electrodes can be connected to a single circuit within the nanopore structure, or each sensor electrode can be connected to its own circuit.

[0088] The array of nanopore structures can be connected into an architecture (sometimes called a pixel) that allows for individual readout from each nanopore structure in the matrix array, and each nanopore structure can have a row number and a column number.

[0089] Each electrical transduction element can have dedicated electronic circuitry, and each electrical transduction element and electronic circuitry can be located within a footprint, which can be a pixel such that the nanopore structures are tessellated within the array.

[0090] While each nanopore structure in the array has an electrical transduction element and, optionally, circuitry and / or control terminals, it will be appreciated in light of the teachings herein that each nanopore structure can have multiple electrical transduction elements and / or multiple circuits, each circuit providing one or more functions. By way of example, a nanopore structure can have an electrical transduction element for sensing and corresponding circuitry for processing signals from the electrical transduction element, and a second electrical transduction element adapted to apply a control signal to a passage within the nanopore structure, the second electrical transduction element having circuitry for controllably applying the control signal.

[0091] Thus, multiple electrical conversion elements can be arranged in a module having multiple respective nanopore structures. The module can have a common dedicated electronic circuit, with each of the electrical conversion elements and electronic circuit being located in the footprint occupied by the multiple nanopore structures. For example, a module can have four nanopore structures, each with its own electrical conversion element, and each electrical conversion element is connected to a common circuit. The common circuit can be addressably connected to an external off-structure or off-sheet electronic circuit.

[0092] The plurality of nanopore structures may be arranged in a two-dimensional matrix. The plurality of nanopore structures may be arranged in a mosaic-like pattern.

[0093] The electrical transduction element may be connected to the base or gate of the transistor for sensing, and the transistor may be a field effect transistor.

[0094] Each nanopore structure may have a control terminal for applying a control signal to vary the potential difference across the respective nanopore structure. The control terminal may be switchably connectable to the electrical transduction element. The control terminal may be switchably connectable to a power source to change a configurable voltage level imposed on the nanopore. The electrical transduction element and the connection for measuring the fluid potential may be switchably isolable from the control signal. The electrical transduction element and the control electrode may be physically separated. At least a portion of the electrical transduction element and at least a portion of the control electrode may extend in the same plane. At least a portion of the electrical transduction element and at least a portion of the control electrode may at least partially form the base of a well. At least a portion of the electrical transduction element and at least a portion of the control electrode may extend perpendicularly from each other. At least a portion of the control electrode may be at least partially configured within a channel. The surface area of ​​the electrical transduction element exposed to the passage may be smaller than the surface area of ​​the control electrode exposed to the passage.

[0095] The devices herein can be configured with a conductive guard configured in at least one of the nanopore layer, the base layer, or the sense layer. To prevent parasitic capacitance from affecting measurements obtained from the connection, the conductive guard can extend between at least one of the electrical transduction element and the signal conductor connected to the electrical transduction element and a parasitic conductive element in the nanopore layer, the base layer, or the sense layer. A buffered version of the input signal can be applied to the guard conductor. As a result, there is no voltage difference across the capacitance from the input signal conductor to the conductive substrate.

[0096] The conductive guard can include an insulating guard conductor having an insulating layer at least partially therein. The conductive guard can be configured to extend at least partially between the base layer and the channel.

[0097] The present inventors have further investigated the operation and manufacturability of the devices disclosed herein.

[0098] In a further aspect, the invention provides a method of operating a device as described for nanopore sensing, the method comprising: translocating an analyte through an array of nanopores under an applied potential difference across the array; measuring a change in fluid potential at each nanopore by a respective electrical transduction element; and, in response to the measurement, applying a control signal to a control terminal of the electrical transduction element to vary the potential difference across the nanopore. Thus, in a further aspect, the present invention provides a method of operating a device for nanopore sensing, the method comprising: imposing a potential difference across an array of nanopore sensors disposed in a structure separating an analyte reservoir and an outlet chamber, each nanopore sensor having a passage for providing fluid connection between the analyte reservoir and the outlet chamber; providing an analyte for analysis by the nanopore sensors, each nanopore sensor having an electrical transducer element for measuring a change in fluid potential at the electrical transducer element of the nanopore sensor when the analyte is guided through a nanopore of the nanopore sensor; and applying a control signal to a control terminal of the electrical transducer element of a nanopore sensor of the array to vary the potential difference across the nanopore sensor. The fluid potential can be measured at the electrical transducer element. The fluid electrical distribution across the nanopore structure can be varied when a fluid is supplied to the device. In operation, a fluid is present in the reservoir, chamber, and passage of the nanopore structure. The fluids in the reservoir, chamber, and passage of the nanopore structure can be different fluids.

[0099] The potential difference imposed across the array serves to direct the analyte through, or at least into, the passageways. The analyte to be analyzed is provided in the analyte reservoir and directed to the outlet chamber, which is accomplished by the drive electrodes. However, this situation can be reversed in that the analyte can be provided to the outlet chamber, or the analyte at the outlet can be directed into the analyte reservoir by the drive electrodes, for example, by changing the potential difference between the drive electrodes.

[0100] In either case, the electrical transduction element of each nanopore structure, with the nanopore acting as a nanopore sensor, can measure changes in fluid potential, and the array of nanopore structures is sized such that the electrical transduction element of one nanopore sensor is inhibited from detecting an analyte passing through a nanopore in an adjacent nanopore structure.

[0101] A control signal can be applied to the element to change the potential difference across the nanopore sensor in which the element resides.

[0102] A control terminal connected to the electrical transducer element can be switchably connected to the control terminal of the electrical transducer element for applying a control signal. Additionally or alternatively, the device can be operated to isolate any sensing circuitry from the electrical transducer element while the control signal is being applied to prevent damage to such circuitry.

[0103] The method can include analyzing a characteristic of the change in electrical potential locally at the nanopore sensor and applying a control signal to the nanopore sensor in response to the predetermined characteristic. The method can apply the control signal to an electrical transduction element of the nanopore sensor to change the electrical potential imposed by a drive electrode at the nanopore sensor. The change in electrical potential difference can induce movement of the analyte, which can be charged, or the freely moving nanopore.

[0104] The control signal can perform multiple actions, including, but not limited to, directing the insertion of a pore into a membrane formed across the passageway, unblocking the nanopore, rejecting the analyte, and altering the rate of movement of the analyte through the nanopore.

[0105] In forming a device having nanopore structures for sensing an analyte, the method of forming includes forming and disposing nanopore structures within a structure to separate an analyte reservoir and an outlet chamber of the device, such that each nanopore structure provides a passage for fluid connection through the structure between the analyte reservoir and the outlet chamber, and fabricating within each nanopore structure an electrical conversion element and electronic circuitry configured to measure a signal from the electrical conversion element, each of the nanopore structures configured to at least one of store, transmit, process, and communicate at least a portion of the signal being measured or information derived from the signal to a connectable processor.

[0106] By fabricating electronic circuits within each nanopore structure, measurements can be made at the electrical transduction element of the nanopore structure when the nanopore is provided to function as a sensor.

[0107] While measurements obtained from sensors can be directly communicated to extra-structure circuitry for analysis, the ability to locally process or condition the signal or information derived from the signal can improve noise performance, data management, or amplification. For example, circuitry located within the nanopore structure can amplify signals received from electrical transduction elements; by amplifying the signal locally, the level of amplified noise is minimized. For example, if signals received from electrical transduction elements are communicated outside the structure before being amplified for analysis, the signal's exposure to noise increases and is subsequently amplified, thus reducing the signal-to-noise ratio.

[0108] The method can further include configuring an analyte reservoir for receiving the analyte and an outlet chamber for collecting the analyte, and configuring a nanopore layer to separate the analyte reservoir and the outlet chamber, which structure allows for separation of the cis and trans channels of the device.

[0109] The method can further include configuring actuation electrodes connected within the analyte reservoir and the exit chamber, respectively, to impose a potential difference across the passage of the nanopore structure. The imposed potential difference can be common across the plurality of nanopore structures. Multiple actuation electrodes can be provided to achieve a common potential difference across the array of nanopore structures.

[0110] The method may further include configuring an electronic circuit with switchable connections to apply signals to respective control terminals of the electrical transduction elements to vary the potential imposed by the drive electrodes across each respective nanopore structure.

[0111] The method may further include forming a control electrode within the passageway of each nanopore sensor, said control electrode being selectively connectable to a signal for varying the potential imposed by the drive electrode across each respective nanopore structure.

[0112] In fabricating a device having nanopore structures for detecting an analyte, the fabrication method includes forming a device having an array of nanopore structures configured in a sheet, wherein the sheet is arranged to separate an analyte reservoir and an outlet chamber of the device so that each nanopore structure provides a passage for fluid connection through the structure between the analyte reservoir and the outlet chamber, the method including: forming a nanopore layer having an array of nanopores and / or an array of support structures such as wells for supporting the nanopores; forming an array of electrical conversion elements; forming a base layer having an array of channels, wherein the base layer is sandwiched or laminated to the nanopore layer to form a sheet so that the nanopores and / or wells are aligned with the electrical conversion elements and channels; and providing a passage through each of the nanopore structures, such that each passage is defined, at least in part, by one of the nanopores and / or one of the wells in the nanopore layer on one side of the passage, the channel in the base layer on the other side of the passage, and the electrical conversion element.

[0113] Aligning the nanopore layer, the base layer, and the array of electrical transduction elements can include sandwiching the array of electrical transduction elements between the nanopore layer and the base layer. The sandwiching step can include bonding or otherwise connecting the two layers.

[0114] The method can further include forming cavities adjacent at least a portion of each of the electrical transduction elements. These cavities can increase the area of ​​the elements exposed to fluid within the passageway.

[0115] The method can further include forming an array of electrical transduction elements on the sense layer and sandwiching the sense layer between the nanopore layer and the base layer.

[0116] The method may further include forming an array of electrical transduction elements on the sense layer; fabricating an array of electronic circuitry in the sense layer, the circuitry connected to each electrical transduction element for modifying and / or processing signals received from the electrical transduction elements; and sandwiching the sense layer between a nanopore layer and a base layer.

[0117] The method may further include arranging the structure to have (i) an exposed portion for connecting to a fluid in the passageway, and (ii) an embedded portion that is embedded within the structure, and / or (iii) a connecting portion for connecting to a measurement circuit that is separate from the structure.

[0118] The method may further include forming a conductive guard in at least one of the nanopore layer, the base layer, or the sense layer, the conductive guard configured to extend between at least one of the electrical transduction element and the signal conductor connected to the electrical transduction element and a parasitic conductive element in the nanopore layer, the base layer, or the sense layer to suppress parasitic capacitance from affecting measurements obtained from the connection.

[0119] The method may further comprise providing a buffer for each nanopore structure, said buffer connecting the output of the electrical transduction element of said nanopore structure to the conductive guard.

[0120] The method can further include providing an amphiphilic membrane to each of the nanopore structures of the array and inserting a biological nanopore into said membrane.

[0121] The method can include removably attaching the structure and / or removing the nanopore layer and replacing it with another nanopore layer, thus allowing the device to be recycled.

[0122] Many embodiments are described herein, and in light of the teachings of this specification, elements of different embodiments can be combined. Thus, many additional embodiments are implicit in light of the teachings of this specification and the drawings, and these often combine two or more of the embodiments described herein. In general, various embodiments can be combined in any combination. (Example 1) 1. A device for nanopore sensing, comprising: a structure disposed to separate an analyte reservoir and an exit chamber, the structure comprising an array of nanopore structures, each nanopore structure comprising a passage for fluid connection through the structure between the analyte reservoir and the exit chamber; a driving electrode connected to the analyte reservoir and the outlet chamber, respectively, to impose a potential difference across the passage; a plurality of electrical transducer elements, each connected to the passage of a respective nanopore structure for measuring fluid potential at the electrical transducer element within the nanopore structure; A device for nanopore sensing comprising: a plurality of control terminals, each connected to a respective nanopore structure for applying a control signal to vary the potential difference across the nanopore structure. (Example 2) The device of Example 1, wherein the electrical conversion element and the control terminal associated with each nanopore structure are directly connected. (Example 3) The device of Example 1 or 2, wherein the terminals are configured to apply a control signal to vary the potential difference across the nanopore structure in response to measuring the fluid potential at the electrical conversion element in each respective nanopore structure. (Example 4) The device of any one of Examples 1 to 3, wherein the application of the control signal is configured to change the potential difference between at least one of the control terminals and at least one of the drive electrodes. (Example 5) A device as described in any one of embodiments 1 to 4, wherein the control signal is connectable to multiple of the nanopore structures to simultaneously vary the potential difference between the connected control terminal and at least one of the driving electrodes. (Example 6) The device of any one of the first to fifth embodiments, wherein the electrical transducer element is isolable from a measurement circuit. (Example 7) The device of Example 6, wherein the electrical transduction element is isolable prior to the application of the control signal. (Example 8) The device of any one of embodiments 1 to 7, wherein the nanopore structure comprises a nanopore. (Example 9) The control signal is and when the device detects that the analyte is blocked, removing the blockage of the passageway of the nanopore. to reject the analyte being measured, and / or to change the direction and / or rate of translocation of an analyte through said nanopore analyte; The device of Example 8, wherein the potential difference across the nanopore is applied to vary the potential difference. (Example 10) A device described in any one of embodiments 1 to 9, wherein the array has electronic circuits, each associated with a respective nanopore structure and connected to the electrical conversion element, and each electronic circuit configured to modify and / or process signals received from the electrical conversion element. (Example 11) The device of Example 10, wherein each electronic circuit is associated with a group of nanopore structures. (Example 12) A device described in any one of embodiments 1 to 11, wherein the array has control circuits, each control circuit associated with a respective nanopore structure and connected to the control terminal and / or the electrical conversion element, and the control circuits configured to vary the potential imposed by the drive electrode at each nanopore structure in response to a signal. (Example 13) The device of Example 12, wherein each control circuit is associated with a group of nanopore structures. (Example 14) The structure is a nanopore layer incorporating nanopores and / or incorporating wells for supporting nanopores; a base layer incorporating the channels; The device of any one of Examples 1 to 13, wherein the nanopore layer and the base layer are sandwiched together such that the nanopores and / or wells align to define the passageways. (Example 15) The device of Example 13, wherein at least one of the electrical conversion element, the control circuit, or the control terminal is disposed on or between the outer surfaces of the structure. (Example 16) A device for sensing an analyte, the device having a plurality of nanopore structures configured within a structure disposed to separate an analyte reservoir and an exit chamber, each nanopore structure providing a passage for fluid communication through the structure between the analyte reservoir and the exit chamber; Each nanopore structure is an electrical conversion element; an electronic circuit configured to detect and optionally amplify a signal from the electrical transduction element; a device, wherein each of said structures is configured to one or more of: store, transmit, process, and communicate at least a portion of said signal to a connectable processor. (Example 17) The device of Example 16, wherein the structure is configured to separate the analyte reservoir for receiving an analyte and the outlet chamber for collecting the analyte. (Example 18) The device of Example 16, wherein each of the nanopore structures within the structure further comprises a compensation circuit. (Example 19) The device of Example 18, wherein the compensation circuit has a variable gain amplifier and / or a variable capacitor in a feedback loop of the compensation circuit. (Example 20) A device described in any one of embodiments 16 to 19, wherein each of the nanopore structures has a control terminal, each control terminal associated with the respective nanopore for applying a control signal to vary the potential difference across the nanopore. (Example 21) The device of Example 20, wherein the control terminal is switchably connected to a power source to vary a configurable voltage level imposed on the nanopore. (Example 22) The device of any one of embodiments 16 to 21, wherein each nanopore structure within the structure is configured into a pixel. (Example 23) The device of Example 22, wherein the pixels form a mosaic array of nanopore structures. (Example 24) 1. A device having an array of nanopore structures configured in a sheet, the sheet comprising: a nanopore layer having an array of nanopores and / or an array of wells for supporting the nanopores; a base layer having an array of channels, said base layer being sandwiched between said nanopore layers to form said sheet, said nanopores and / or said wells being aligned with said channels, each of said nanopore structures comprising a passageway, each passageway being at least partially one of the nanopores and / or one of the wells in the nanopore layer on one side of the passage; a channel in the substrate on the other side of the passage; an electrical transduction element; and (Example 25) A device as described in Example 24, wherein the electrical conversion element in each passage is positioned between the nanopore layer and at least a portion of the channel and configured with a connection for measuring the electrical potential of the fluid at the position of the electrical conversion element when fluid is supplied into the passage. (Example 26) The device described in Example 24 or 25, further comprising an analyte reservoir and an outlet chamber at least partially separated by the sheet, the sheet having an array of passages disposed between the analyte reservoir and the outlet chamber to connect the analyte reservoir to the outlet chamber. (Example 27) The device of any one of Examples 24 to 26, further comprising a driving electrode connected to the analyte reservoir and the outlet chamber to impose a potential difference across the array of passages between the analyte reservoir and the outlet chamber. (Example 28) A device as described in any one of embodiments 24 to 27, wherein the sheet is substantially planar and has a cis surface on the nanopore layer facing an analyte reservoir and defining a cis surface, and a trans surface on the base layer facing an exit chamber and defining a trans surface, and the electrical conversion elements of the array are at least partially embedded within the sheet between the cis surface and the trans surface. (Example 29) The device of any one of Examples 24 to 28, wherein the electrical transducer elements of the array are sandwiched between the nanopore layer and the base layer. (Example 30) A device described in any one of embodiments 24 to 29, wherein each nanopore structure of the array has a well formed at a first end of the passage, a nanopore configured at the first end of the well, and the electrical conversion element configured on the opposite side of the well from the nanopore. (Example 31) The device of Example 30, wherein the well is configured to support a fluid membrane, such as a polymer membrane or a lipid bilayer. (Example 32) The device of any one of Examples 24 to 31, wherein the nanopore layer is made of a different material than the base layer. (Example 33) A device described in any one of embodiments 24 to 32, wherein the electrical conversion element is a sensor electrode that can be directly connected to the base or gate of a transistor device to measure fluctuations in the electrical potential of the fluid at the position of the electrical conversion element when the fluid is supplied into the passage. (Example 34) The device of any one of Examples 24 to 33, wherein each electrical transduction element is connected, optionally through vias, to an edge connector or wire bond to measurement circuitry off the sheet. (Example 35) The device of Example 33, wherein the transistor device is a field effect transistor. (Example 36) The device of any one of embodiments 24 to 35, further comprising a sense layer having the array of electrical conversion elements, the sense layer being sandwiched between the nanopore layer and the base layer. (Example 37) The device of Example 36, wherein the electrical conversion element is formed on the sense layer, and the electrical conversion element has (i) an exposed portion for connecting to fluid in the passage, and (ii) an embedded portion embedded in the sheet, and / or (iii) a connection portion for connecting to a measurement circuit separate from the sheet. (Example 38) The device of any one of Examples 24 to 37, wherein the electrical conversion element at least partially covers a wall of the passageway. (Example 39) The device of any one of embodiments 24 to 38, wherein the electrical conversion element, in cross section, covers a portion of the wall of the channel. (Example 40) The device of any one of the preceding embodiments, wherein the electrical conversion element forms an annulus around the passageway. (Example 41) A device described in any one of embodiments 24 to 40, further comprising an analyte reservoir, wherein the electrical conversion element at least partially forms a surface of the base layer or the sense layer around the passage and has an exposed portion arranged to face the analyte reservoir. (Example 42) 42. The device of any one of Examples 24 to 41, further comprising an outlet chamber, wherein the electrical conversion element at least partially forms a surface of the sense layer around the passage and has an exposed portion disposed facing the outlet chamber. (Example 43) 43. The device of Example 42, wherein the exposed portion forms part of a wall of a cavity formed in the sense layer between the well and the channel. (Example 44) The device of any one of embodiments 24 to 43, wherein the electrical conversion element has an opening that forms a portion of the passage and the exposed portion, and in cross section, the ratio of the size of the exposed portion of the electrical conversion element to the size of the opening is 1:1. (Example 45) The device of Example 44, wherein the ratio is about 5:1. (Example 46) The device of any one of embodiments 24 to 45, wherein the electrical conversion element has an opening that forms part of the passage and the exposed portion, and in a planar view, the ratio of the size of the exposed portion of the electrical conversion element to the size of the opening is 1:1. (Example 47) The device of Example 46, wherein the electrical conversion element has an opening that forms a portion of the passage and the exposed portion, and the ratio is approximately 5:1. (Example 48) The device of any one of embodiments 44 to 47, wherein the opening is circular. (Example 49) A device described in any one of embodiments 36 to 48, wherein the sense layer incorporates electronic circuitry connected to the electrical conversion element for modifying and / or processing the signal received from the electrical conversion element. (Example 50) A device as described in Example 49, wherein the electronic circuit is configured to detect changes in the resistance of the nanopore in each passage when an analyte passes through the nanopore, and the changes in resistance are detected through changes in fluid potential at the electrical conversion element of the sensor. (Example 51) The device of example 49 or 50, wherein the electronic circuit determines a change in potential in the electrical conversion element when the polymer passes through the nanopore, converts the change in potential into a voltage signal, and amplifies the voltage signal. (Example 52) 52. The device of any one of embodiments 49 to 51, wherein the electronic circuit filters the signal. (Example 53) 53. The device of any one of embodiments 49 to 52, wherein the electronic circuitry samples and / or digitizes the signal obtained from the electrical conversion element. (Example 54) The device of any one of embodiments 49 to 53, wherein each nanopore structure has multiple electrical conversion elements. (Example 55) A device described in any one of embodiments 49 to 54, wherein each nanopore structure in the array has a plurality of electrical conversion elements corresponding to each nanopore, and the electrical conversion elements are arranged in an addressable array. (Example 56) A device described in any one of embodiments 49 to 55, wherein each electrical conversion element has a dedicated electronic circuit, each electrical conversion element and electronic circuit is arranged within a footprint, and the footprints are tessellated within the array. (Example 57) A device described in any one of embodiments 49 to 56, wherein multiple electrical conversion elements are arranged in a module having multiple nanopore structures, the module has a common dedicated electronic circuit, each of the electrical conversion elements and electronic circuits is arranged within a footprint, and the footprints are mosaicked within the array. (Example 58) The device of any one of embodiments 1 to 57, wherein the plurality of nanopore structures are arranged in a two-dimensional matrix. (Example 59) The device of any one of embodiments 1 to 58, wherein the plurality of nanopore structures are arranged in a mosaic pattern. (Example 60) The device of any one of embodiments 1 to 59, wherein the electrical transducer is connected to the base or gate of a transistor for sensing. (Example 61) A device described in any one of embodiments 24 to 60, wherein each of the nanopore structures has a control terminal for applying a control signal to vary the potential difference across the respective nanopore structure. (Example 62) The device of Example 61, wherein the control terminal is switchably connectable to the electrical conversion element. (Example 63) The device of Example 62, wherein the control terminal is switchably connectable to a power source to change the configurable voltage level imposed on the nanopore. (Example 64) A device described in any one of embodiments 61 to 63, wherein the electrical conversion element and the connection for measuring the potential of the fluid are switchably isolable from the control signal. (Example 65) The device of any one of embodiments 61 to 64, wherein the electrical conversion element and the control electrode are physically separated. (Example 66) The device of Example 65, wherein at least a portion of the electrical conversion element and at least a portion of the control electrode extend in the same plane. (Example 67) The device of example 65 or 66, wherein at least a portion of the electrical conversion element and at least a portion of the control electrode at least partially form a base of a well. (Example 68) The device of Example 65, wherein at least a portion of the electrical conversion element and at least a portion of the control electrode extend perpendicularly from each other. (Example 69) The device of any one of embodiments 65 to 68, wherein at least a portion of the control electrode is configured at least partially within the channel. (Example 70) The device of any one of embodiments 65 to 69, wherein the surface area of ​​the electrical conversion element exposed to the passage is smaller than the surface area of ​​the control electrode exposed to the passage. (Example 71) Example 2. Further comprising: a conductive guard configured to extend between at least one of the electrical transduction element and a signal conductor connected to the electrical transduction element and a parasitic conductive element within the nanopore layer or base layer to suppress parasitic capacitance from affecting measurements obtained from the connection within at least one of the nanopore layer or base layer. The device described in 4 to 70. (Example 72) A device as described in any one of Examples 36 to 71, wherein a conductive guard is additionally or alternatively configured to extend between at least one of the electrical conversion element and a signal conductor connected to the electrical conversion element and a parasitic conductive element in the sense layer to suppress parasitic capacitance from affecting measurements obtained from the connection within the sense layer. (Example 73) 73. The device of Example 71 or 72, wherein the conductive guard comprises, at least in part, a guard conductor and an insulating layer configured to insulate the guard conductor from the guarded conductor or conductors being guarded. (Example 74) 74. The device of any one of embodiments 71 to 73, wherein the conductive guard is configured to extend at least partially between the base layer and the channel. (Example 75) 1. A method of operating a device for nanopore sensing, comprising: imposing a potential difference across an array of nanopore sensors disposed within a structure separating an analyte reservoir and an exit chamber, each nanopore sensor having a passage for providing a fluid connection between the analyte reservoir and the exit chamber; providing an analyte for analysis by the nanopore sensors, each nanopore sensor having an electrical transduction element for measuring a change in electrical potential across the electrical transduction element of the nanopore sensor when an analyte is guided through a nanopore of the nanopore sensor; applying control signals to control terminals of electrical transduction elements of nanopore sensors of the array to vary the potential difference across the nanopore sensors. (Example 76) 76. The method of example 75, further comprising switchably connecting the control terminal to the electrical transducer element and applying the control signal to the control terminal. (Example 77) The method of example 75 or 76, further comprising analyzing characteristics of the change in fluid potential at the electrical transducer element of the nanopore sensor, and applying the control signal to the nanopore sensor in response to at least one of the characteristics. (Example 78) 78. The method of any one of embodiments 75-77, wherein applying a control signal to an electrical transducer element of a nanopore sensor changes the potential imposed by the drive electrode at the nanopore sensor. (Example 79) 79. The method of any one of Examples 75 to 78, further comprising isolating the electrical transducer element from a measurement circuit connected to the nanopore sensor before applying the control signal. (Example 80) The nanopore sensor further includes applying the control signal to the nanopore sensor in response to measuring a change in fluid potential at the electrical transduction element of the nanopore sensor, the control signal comprising: removing the blockage of the nanopore when the blockage is detected; rejecting the analyte being measured by the nanopore sensor; and / or The method of any one of embodiments 75 to 79, wherein the method is configured to change the rate and / or direction of movement of the analyte through the nanopore. (Example 81) 1. A method of forming a device having a nanopore structure for sensing an analyte, comprising: forming nanopore structures within the structure and disposing the structures to separate an analyte reservoir and an exit chamber of the device, such that each nanopore structure provides a passage for fluid connection through the structure between the analyte reservoir and the exit chamber; Within each nanopore structure, an electrical conversion element; and an electronic circuit configured to measure a signal from the electrical transduction element; wherein each of the nanopore structures is configured to at least one of store, transmit, process, and communicate at least a portion of the signal being measured or information derived from the signal to a connectable processor. (Example 82) The method of Example 81, further comprising configuring an analyte reservoir for receiving an analyte and an outlet chamber for collecting the analyte, and configuring the nanopore layer to separate the analyte reservoir and the outlet chamber. (Example 83) The method of Example 82, further comprising configuring driving electrodes connected within the analyte reservoir and the outlet chamber, respectively, to impose a potential difference across the passage of the nanopore structure. (Example 84) The method of Example 83, wherein the imposed potential difference is common across multiple of the nanopore structures. (Example 85) The method of Example 85, further comprising configuring the electronic circuit with a switchable connection to apply a signal to each control terminal of the electrical conversion element to vary the potential imposed by the drive electrode across each respective nanopore structure. (Example 86) The method of any one of Examples 81 to 84, further comprising forming a control electrode within the passage of each nanopore sensor, the control electrode being selectively connectable to a signal for varying the potential imposed by the drive electrode across each respective nanopore structure. (Example 87) 1. A method of forming a device having an array of nanopore structures configured in a sheet, the method comprising: disposing the sheet to separate an analyte reservoir and an outlet chamber of the device such that each nanopore structure provides a passage for fluid connection through the structure between the analyte reservoir and the outlet chamber; forming a nanopore layer having an array of nanopores and / or an array of wells for supporting the nanopores; forming an array of electrical transduction elements; forming a base layer having an array of channels, the base layer being sandwiched between the nanopore layer to form the sheet such that the nanopores and / or the wells are aligned with the electrical transduction elements and channels; providing a passageway through each of said nanopore structures, so that each passageway is at least partially comprised of: one of the nanopores and / or one of the wells in the nanopore layer on one side of the passage; a channel in the substrate on the other side of the passage; an electrical conversion element; and (Example 88) The method of Example 87, wherein aligning the nanopore layer, base layer, and array of electrical conversion elements comprises sandwiching the array of electrical conversion elements between the nanopore layer and the base layer. (Example 89) 89. The method of any one of embodiments 87 and 88, further comprising forming a cavity adjacent at least a portion of each of the electrical transduction elements. (Example 90) The method of any one of embodiments 87 to 89, further comprising forming an array of the electrical conversion elements on a sense layer and sandwiching the sense layer between the nanopore layer and the base layer. (Example 91) 90. The method of any one of embodiments 87-89, further comprising: forming an array of the electrical transducer elements on a sense layer; fabricating an array of electronic circuitry in the sense layer, the circuitry connected to each electrical transducer element for modifying and / or processing the signal received from the electrical transducer element; and sandwiching the sense layer between the nanopore layer and the base layer. (Example 92) The electrical conversion element (i) an exposed portion for connecting to a fluid in said passageway; and (ii) an embedded moiety embedded within said structure; and / or (iii) a connection portion for connecting to a measurement circuit separate from the structure, Law. (Example 93) 93. The method of any one of embodiments 87 to 92, further comprising forming a conductive guard in at least one of the nanopore layer, base layer, or sense layer, the conductive guard configured to extend between the electrical conversion element and at least one of the signal conductors connected to the electrical conversion element and a parasitic conductive element in the nanopore layer, base layer, or sense layer to suppress parasitic capacitance from affecting measurements obtained from the connection. (Example 94) 93. The method of any one of the embodiments to 93, further comprising providing a buffer for each nanopore structure, the buffer connecting the output of the electrical conversion element of the nanopore structure to a conductive guard. (Example 95) The method of any one of embodiments 81 to 94, further comprising providing an amphiphilic membrane to each of the nanopore structures of the array and inserting a biological nanopore into the membrane. (Example 96) The method of any one of embodiments 81 to 95, wherein the nanopore layer is removably attachable to the structure, and the method further comprises removing the nanopore layer and replacing the nanopore layer with another nanopore layer.

[0123] Embodiments of the invention will now be discussed, by way of non-limiting example only, with reference to the drawings in which: [Brief explanation of the drawings]

[0124] [Figure 1] 1 is a cross-sectional view of a known nanopore sensor. [Figure 2] FIG. 1 is a cross-sectional view of a single sensor electrode and a corresponding biological nanopore in a nanopore array configured in the structure and connected via wires to measurement circuitry. [Figure 3(a)] An alternative cross-sectional view of a single sensor electrode and corresponding biological nanopore, where the sensor electrode is configured on a sensor layer sandwiched between a nanopore layer and a base layer within a portion of a nanopore structure array, and the sensor electrode is connected to an electronic circuit via a wire. [Figure 3(b)] FIG. 3( a ) is a view corresponding to FIG. 3( a ), in which the sense layer incorporates electronic circuitry. [Figure 4(a)] FIG. 3(b) is a schematic diagram of the layout of the nanopore sensor of FIG. 3(b), showing the location of the wells relative to the electronic circuitry. [Figure 4(b)] FIG. 1 shows two adjacent sensor electrodes before the addition of a nanopore. [Figure 4(c)] FIG. 4(b) shows an example of a portion of a structure having an array of nanopore sensors of FIG. 4(a). [Figure 4(d)] FIG. 4(b) shows an example of a portion of a structure having an array of nanopore sensors of FIG. 4(a). [Figure 4(e)] FIG. 1 is a schematic diagram showing how structures can be arranged to separate two chambers in a device. [Figure 4(f)] FIG. 1 shows an alternative layout of four nanopore structures. [Figure 5(a)] 1 illustrates various electronic circuits that implement the electronic functions that interface with the electrical transducer elements. [Figure 5(b)] 1 illustrates various electronic circuits that implement the electronic functions that interface with the electrical transducer elements. [Figure 5(c)] 1 illustrates various electronic circuits that implement the electronic functions that interface with the electrical transducer elements. [Figure 5(d)] 1 illustrates various electronic circuits that implement the electronic functions that interface with the electrical transducer elements. [Figure 6(a)] 1 is a cross-sectional schematic diagram of sensor and control electrodes configured in an array of nanopore sensors. [Figure 6(b)] 1 is a cross-sectional schematic diagram of sensor and control electrodes configured in an array of nanopore sensors. [Figure 6(c)] 1 is a cross-sectional schematic diagram of sensor and control electrodes configured in an array of nanopore sensors. [Figure 6(d)] 1 is a cross-sectional schematic diagram of sensor and control electrodes configured in an array of nanopore sensors. [Figure 6(e)]1 is a cross-sectional schematic diagram of sensor and control electrodes configured in an array of nanopore sensors. [Figure 7(a)] 1A and 1B show two schematic circuits illustrating the parasitic capacitance of an array, with and without guarding, respectively. [Figure 7(b)] 3 is an alternative cross-sectional view to that shown in FIG. 2, in which a guard conductor is configured within the structure and connected to the measurement circuitry via an additional wire. [Figure 7(c)] 10A and 10B are cross-sectional views of alternative guard configurations; [Figure 7(d)] 10A and 10B are cross-sectional views of alternative guard configurations; [Figure 8] 1A-1C are side views of respective examples of devices containing an array of nanopore structures. [Figure 9] 1A-1C are side views of respective examples of devices containing an array of nanopore structures. DETAILED DESCRIPTION OF THE INVENTION

[0125] The embodiments typically include corresponding components labeled with common reference numerals. For clarity, descriptions of corresponding components are not repeated but generally apply to all embodiments unless the context otherwise requires. Not every component is labeled in every figure, and not every component of every embodiment of the invention is shown unless illustration is necessary to understand the invention.

[0126] In the overview, a device for improved nanopore sensing is described. An exemplary device can have a structure arranged to separate an analyte reservoir and an exit chamber. The structure can have an array of nanopore structures, each nanopore structure comprising a passage for fluid connection through the structure between the analyte reservoir and the exit chamber. Control terminals can be included, each control terminal connecting to a respective nanopore structure for applying a control signal to vary the potential difference across the nanopore structure. In a further embodiment, the improved nanopore structure for analyte sensing can include electronic circuitry configured to detect a signal from an electrical transduction element, and each nanopore structure can be configured to at least one of store, transmit, process, and communicate at least a portion of the signal to a processor.

[0127] Some embodiments of devices for improved nanopore sensing have an array of nanopore structures configured in a sheet, the sheet comprising a nanopore layer having an array of nanopores and / or an array of wells for supporting the nanopores, and a base layer having an array of channels, the base layer being sandwiched or laminated to the nanopore layer to form the sheet, the nanopores and / or wells being aligned with the channels, and each of the nanopore structures comprising a passage, each passage being defined at least in part by one of the nanopores and / or one of the wells in the nanopore layer on one side of the passage, the channel in the base layer on the other side of the passage, and an electrical transduction element.

[0128] Aspects of the present invention further relate to a method of operating a device for nanopore sensing, the method comprising applying control signals to control terminals of electrical conversion elements of nanopore structures of an array to vary the potential difference across the nanopore sensor.

[0129] Additional embodiments relate to methods of forming devices having nanopore structures for sensing analytes. An exemplary method can include fabricating, in each nanopore structure, an electrical transduction element and an electronic circuit configured to measure a signal from the electrical transduction element.

[0130] 2-4(a) are cross-sectional views of a portion of a structure 100 incorporating a nanopore structure. The structure 100 has an array of nanopore structures, each adapted to support a nanopore 116. The nanopore structures of the device, when configured with a nanopore, can function as a nanopore sensor. The nanopore sensor 102 herein is a nanopore structure having a nanopore.

[0131] Figures 4(b)-4(f) illustrate that multiple nanopore sensors 102 shown in Figures 2-4(a) may be arranged as part of an array of nanopore structures 104. Such an arrangement may be referred to as a two-dimensional matrix of nanopore structures, or an array of nanopore sensors.

[0132] The structure 100, which can take the form of a sheet, incorporates an array of nanopore structures 104 (note that only one nanopore sensor 102 of the array is shown), and can be configured into a device, as shown in Figure 4(e), or a device for analyzing an analyte.

[0133] The structure 100 separates an analyte reservoir 106 for receiving an analyte and an outlet chamber 108. The structure 100 has a nanopore layer 110 configured on a base layer 112, which together form at least a portion of the structure 100 with a plurality of nanopore sensors 102. Each nanopore sensor 102 in the array 104 has a passageway 114 or fluid passageway configured to extend through the nanopore layer 110 and base layer 112 of the array 104 to connect the analyte reservoir 106 and the outlet chamber 108. The analyte reservoir 106 may also be known as an analyte chamber, sample chamber, cis, cis reservoir, or first fluid reservoir. The outlet chamber may also be known as a trans, trans reservoir, or second fluid reservoir.

[0134] The nanopore layer 110 of each nanopore sensor 102 may optionally include a nanopore 116 in a membrane 118 supported by the nanopore layer 110. Alternatively, the nanopore 116 may be a so-called solid-state nanopore, i.e., a nanometer-sized through-hole provided in a solid support layer. Alternatively, the nanopore 116 may be a so-called hybrid nanopore, i.e., a biological nanopore provided in an opening in a solid membrane. In either case, the nanopore 116 is provided in the membrane 118 proximal to the first end 120 of the passageway 114 or the pore end (e.g., at the top of the sensor as shown).

[0135] The base layer 112 has a second end 124, which is the opposite end of the passage 114 relative to the first end 120, or a channel 122 proximal to the channel end (e.g., at the bottom of the illustrated nanopore sensor 102). The passage 114 extends through the nanopore structure 100 connecting one side to the other. The channel 122 forms a portion of the passage 114. The channel 122 is structurally and geometrically configured to function as a fluidic resistor. This can be achieved by defining the aspect ratio of the channel 122. Additionally or alternatively, other techniques for implementing fluidic resistance in the channel 122 may be used.

[0136] The fluidic resistance of channel 122 can be varied by changing its dimensions, particularly its aspect ratio, as well as by changing the ionic concentrations of the fluids in analyte reservoir 106 and outlet chamber 108. For example, channel 122 can be configured with a high aspect ratio to increase the resistance. Additionally or alternatively, the fluid in channel 122 can have a lower ionic concentration compared to the fluids in analyte reservoir 106 and outlet chamber 108 to increase the resistance of the channel. Maintaining a higher ionic concentration in analyte reservoir 106 and outlet chamber 108 improves the signal-to-noise ratio.

[0137] In some embodiments, the aspect ratio, which is the ratio of channel length to channel diameter or largest lateral dimension, can be, for example, from about 100:1 to about 2000:1.

[0138] In some embodiments, the ion concentration difference, which is the ratio of the ion concentration in the analyte reservoir 106 and / or outlet chamber 108 to the ion concentration in the channel 122, can be from about 1:1 to about 2000:1, for example, about 1000:1.

[0139] Channel 122 can be configured such that when passageway 114 is occupied by fluid, the resistance of channel 122 substantially matches the resistance of nanopore 116 and is relatively high compared to the resistance of the fluid in analyte reservoir 106 and outlet chamber 108, so that the resistances of analyte reservoir 106 and outlet chamber 108 do not appreciably affect the measurement. In other words, channel 122 is configured as a fluidic resistor to approximate the resistance of nanopore 116. This means that the resistances of other circuit elements, such as the fluid in analyte reservoir 106 and outlet chamber 108, are less significant and do not require compensation to account for them when measurements are made.

[0140] The signal-to-noise ratio can be optimized by selecting the fluidic resistance of channel 122 to be equal to the resistance of nanopore 116. However, this is not required, and the fluidic resistance of channel 122 can be varied from this value to take other factors into account while still obtaining an acceptable signal-to-noise ratio. An acceptable signal-to-noise ratio can be achieved, for example, by making the fluidic resistance of channel 122 significantly less than the resistance of nanopore 116, e.g., making the fluidic resistance of channel 122 no greater than 10% of the resistance of nanopore 116, e.g., no greater than 2% of the resistance of nanopore 116. In some embodiments, the lower limit of the fluidic resistance of channel 122 can be set by the desired signal-to-noise ratio. In other embodiments, the lower limit of the fluidic resistance of channel 122 can be set by the threshold for crosstalk between adjacent channels during flicking (as described below). That is, the fluid resistance of the channels 122 is desirably significantly greater than the resistance from the ends of the channels 122 to the electrical transduction elements to prevent these resistances from forming a voltage divider that applies a portion of the applied voltage to adjacent channels 122.

[0141] Other factors that may be considered in selecting the fluid resistance of the channel 122 are as follows.

[0142] As the fluidic resistance of channel 122 increases, ion diffusion decreases and ion depletion near the pore increases, causing signal attenuation over the timescale of a typical event for which a signal is obtained. To increase the read length limit caused by this effect, the fluidic resistance of channel 122 can be reduced. In many embodiments, this factor can place an upper limit on the fluidic resistance of channel 122.

[0143] Because the channel 122 and nanopore 116 act as a voltage divider, the voltage across the nanopore 116 is affected by the current flowing through it. As the fluidic resistance of the channel 122 increases, the fluctuations in the voltage across the nanopore 116 increase, which can complicate signal processing. To limit this effect, the fluidic resistance of the channel 122 can be reduced.

[0144] Channels with lower fluidic resistance are easier to fabricate and may open up alternative fabrication techniques that improve yield or reduce cost.

[0145] Reducing the fluid resistance of the channel 122 can increase the bandwidth or provide room for additional capacitance in the passage 114 .

[0146] Taking these factors into consideration, the fluidic resistance of channel 122 may be less than the resistance of nanopore 116, such as typically up to 50%, or up to 25% of the resistance of nanopore 116. In some embodiments, the optimal fluidic resistance of channel 122 may be about 10% of the resistance of nanopore 116.

[0147] When the ratio of the fluidic resistance of the channel 122 to the resistance of the nanopore 116 is reduced, the signal-to-noise ratio is not directly proportional to the resistance ratio. For example, in some embodiments, when the fluidic resistance of the channel 122 is about 10% of the resistance of the nanopore 116, the signal-to-noise ratio is about 30% of its optimal value.

[0148] The channel 122 can be formed in the wafer, and after the passage 114 is formed therethrough, an oxide layer can be used to reduce the diameter of the passage 114 through the base layer 112, thus allowing the amount of oxidation to adjust the aspect ratio.

[0149] A sensor electrode 126, or sensor element, is disposed between the nanopore 116 and at least a portion of the channel 122. The sensor electrode 126 forms an electrical transduction element in this example. More generally, the sensor electrode 126 may be adapted to form any of the various types of electrical transduction elements disclosed in WO2016 / 127007.

[0150] The sensor electrode 126 is at least partially exposed to the passageway 114 in the nanopore sensor 102 and is configured with a connection 128 for measuring the electrical potential of the fluid at the location of the sensor electrode 126 when the fluid is supplied into the passageway 114. Together with the nanopore layer 110 and the base layer 112, the sensor electrode 126 defines a wall of the passageway 114. The connection 128 may be a wire bond to a separate electronic circuit 130, which is configured to analyze the signal obtained from the sensor electrode 126.

[0151] The analyte reservoir 106 can function as a first fluid reservoir, while the outlet chamber 108 can function as a second fluid reservoir. The structure 100 can at least partially separate the analyte reservoir 106 and the outlet chamber 108, with the passage 114 of the sensor 102 connecting the analyte reservoir 106 to the outlet chamber 108.

[0152] During use, the passageway 114 of each nanopore sensor 102 is occupied by a fluid. Additionally, the actuation electrodes 132 in the analyte reservoirs 106 and outlet chambers 108 include at least one respective cis electrode 132 a, also known as an analyte electrode, and at least one respective trans electrode 132 b, also known as an outlet electrode, that are configured to impose a potential difference across the passageway 114 of the nanopore sensors 102 in the array 104 between the analyte reservoirs 106 and the outlet chambers 108.

[0153] The structure 100 can be substantially planar. The array 104 can be substantially planar. Non-planar configurations are contemplated by the inventors but are not described herein. The nanopore sensors 102 in the array 104 have a cis surface 134 of the nanopore layer 110 facing the analyte reservoir 106 and disposed to define a cis face 136, and a trans surface 138 of the base layer 112 facing the exit chamber 108 to define a trans face 140. The cis face 136 and the trans face 140 are shown by dashed lines in FIGS. 2, 3(b), and 7(b). The sensor electrodes 126 are embedded within the structure 100 between the cis face 136 and the trans face 140. The nanopore 116 can be located substantially on the cis-face 136 of the first end 120 of the passage 114, while the second end 124 of the passage 114 can be located substantially on the trans-face 140.

[0154] 2, the sensor electrode 126 may be at least partially embedded within the structure 100 between the nanopore layer 110 and the base layer 112. In other words, the sensor electrode 126 is sandwiched or laminated between the nanopore layer 110 and the base layer 112.

[0155] The nanopore layer 110 has a well 142 formed at the first end 120 of the passage 114. In the example of FIG. 2, the nanopore 116 is configured at the first end 120 of the passage 114, substantially on the cis-face 136, on one side of the well 142. The sensor electrode 126 is configured on the opposite side of the well 142 from the nanopore 116, as shown. The well 142 is shown as a cup-shaped depression with a membrane 118 across its edge, shown in cross-section. The well 142 is configured to receive an analyte that has passed through the nanopore 116. Note that the well 142 is fluidly connected to the channel 122 via a well opening 142a, which can be described as a well outlet. The well opening 142a functions to allow the analyte chamber to be fluidly connected to the outlet chamber. The well opening 142a does not function as a nanopore. In some implementations, the well opening 142a is configured to allow the analyte to pass therethrough unimpeded, i.e., without affecting the movement of the analyte from the analyte reservoir 106 to the outlet chamber 108.

[0156] Well opening 142a provides a fluid connection between analyte reservoir 106 and outlet chamber 108, while allowing analyte that passes through nanopore 116 to remain within well 142. Well 142 and channel 122 can be considered part of outlet chamber 108. In the example shown in FIG. 2, well opening 142a is located in the center of the base of well 142 within sensor electrode 126.

[0157] The well 142, or more generally the nanopore layer 110, is configured to support a membrane 118, such as a polymer membrane or lipid bilayer, sometimes referred to as a fluid membrane. The nanopore layer 110 can be fabricated separately from the base layer 112. The nanopore layer 110 can be formed from a different material than the base layer 112. The nanopore layer 110 can be formed from at least one of a photolithographically prepared material, a molded polymer, or a laser-etched plastic.

[0158] In some embodiments, the sensor electrode 126 can be directly connected to the base or gate of a sense transistor 153 (shown in FIG. 5( a) and described further below) to measure the potential of the fluid at the location of the sensor electrode 126 when fluid is provided in the passageway 114. In some cases, the sensor electrode 126 can be connected to an edge connector or wirebond, and optionally by conductive vias and / or interconnects, to an electronic circuit 130, which is an example of a measurement circuit outside the structure. The sense transistor 153 can be a field-effect transistor (FET), and the configuration of the sense transistor 153 and its optional integration into the structure 100 are described in the examples below. In some embodiments, the sense transistor 153 (not shown) can be located within the electronic circuit 130.

[0159] The nanopore sensor 102 shown in Figure 2 is an example in which the sensor electrode 126 can be formed on the base layer 112. The sensor electrode 126 in Figure 2 can be formed directly on the base layer 112, or alternatively, can be formed separately on the sense layer 144, as shown in Figures 3(a) and 3(b). After forming the sensor electrode 126 on the sense layer 144, the sense layer 144 can be sandwiched between the nanopore layer 110 and the base layer 112, resulting in the structure shown in Figure 3(a).

[0160] The sense layer 144 can be fabricated in a manner similar to the base layer 112, with the wafer having passages 114 formed therethrough substantially perpendicular to the surface of the wafer. Alternatively, the wafer can be post-processed to open the passages 114. The passages 114 and / or channels 122 can be formed using techniques such as photolithography or deep reactive ion etching (DRIE), or a combination thereof. The wafer can be surrounded by an oxide layer. The wafer can be a CMOS wafer. Sensor electrodes 126 can be formed on one side of the sense layer 144 around the passages 114 on the sense layer 144. The passages 114 through the sense layer 144 and the sensor electrodes 126 formed around these passages 114 are arranged to have a pitch or layout that results in alignment with the channels 122 on the base layer 112. When secured together, the passages 114 in the sense layer 144 are aligned with the channels 122 in the base layer 112.

[0161] By way of example, the nanopore layer 110 can be made from a polymer that can be molded or lithographically etched, the base layer 112 can be formed from a silicon wafer, and / or the sense layer 144 can be a CMOS wafer.

[0162] The sense layer 144 can be aligned and bonded to the base layer 112 in one of two orientations. In one orientation (not shown), the sensor electrode 126 remains fully exposed after bonding. That is, the sensor electrode 126 is not sandwiched between the sense layer 144, but is distal from the base layer 112 after the sense layer 144 is secured to the base layer 112, and is subsequently sandwiched between the sense layer 144 and the nanopore layer 110. In the other orientation, as shown in FIG. 3( a), the sensor electrode 126 is formed on top of the sense layer 144, which is then inverted and then bonded to the base layer 112 so that the sensor electrode 126 faces downward and is sandwiched between the sense layer 144 and the base layer 112, as can be seen. Prior to bonding in this configuration, a portion of the oxide layer on the base layer 112 around the channel 122 can be etched away or otherwise removed to create a cavity 146 such that after bonding, an increased area of ​​the sensor electrode 126 is exposed to the passageway 114. The area of ​​the exposed sensor electrode 126 can be maximized to increase contact with the fluid in the passageway 114.

[0163] The wells 142 in the nanopore layer 110 are aligned with the passages 114 and the sensor electrodes 126 and are bonded to the sense layer 144 with the sensor electrodes 126 sandwiched in between. Looking at FIG. 3(b) and noting that the sense layer 144 is fabricated from the bottom up, the final step is to deposit the sensor electrodes 126 on top. Once assembled, the sense layer 144 is flipped over so that the sensor electrodes 126 that were on top are now facing down, as shown in FIG. 3(b). The cavities 146 etched from the oxide layer (grey) of the base layer 112 mean that the sensor electrodes 126 are fully exposed.

[0164] The sensor electrode 126 remains at least partially exposed to the passageway 114 and is configured with connections for measuring the electrical potential of the fluid at the location of the sensor electrode 126 in the nanopore 116 as the fluid is delivered into the passageway 114. Arrangements of the sensor electrode 126 that minimize its surface area openly facing one of the analyte reservoir 106 or the outlet chamber 108 (e.g., the arrangements of FIG. 2 or FIG. 3(a)) serve to limit exposure to the analyte reservoir 106 or the outlet chamber 108 and inhibit contamination of the surface of the sensor electrode 126. One such example is shown in FIG. 3(a), which shows the sensor electrode 126 substantially partially surrounded by the passageway 114. Prior to fluid delivery or during formation of the amphiphilic membrane to support the biological nanopore, the surface of the sensor electrode 126 may be exposed to fluids that could contaminate the surface of the sensor electrode 126, thus mitigating any risk of contamination.

[0165] In one configuration, at least a portion of the sensor electrode 126 can be disposed to face outward from the well 142 toward the channel 122, as shown in FIG. 3( a). The exposed portion of the sensor electrode 126 provides a connection to the fluid in the passageway 114 for detecting fluctuations in fluid potential at the sensor electrode 126 as the analyte passes through the nanopore 116. The sensor electrode 126 can also have an embedded portion embedded within the structure 100. The sensor electrode 126 can also have a connection 128, such as a wire bond, for connection to electronic circuitry 130, such as measurement or control circuitry, which can be separate from the structure 100, as shown in FIG. 3( a).

[0166] In each of the embodiments, the sensor electrodes 126 can be configured in various configurations for exposure to fluid within the passageway 114, and can at least partially cover the walls of the passageway 114, cover a portion of the walls of the passageway 114 in cross section, form an annulus around the passageway 114, at least partially form a surface of the base layer 112 or sense layer 144 around the passageway 114 with an exposed portion disposed facing the analyte reservoir 106, or at least partially form a surface of the sense layer 144 around the passageway 114 with an exposed portion disposed facing the outlet chamber 108. In particular, a cavity 146 can be formed in the passageway 114 to create an area that increases the area of ​​exposure of the sensor electrodes 126 and allows them to contact an increased amount of fluid. The cavity 146 is formed by a recess formed in the base layer 112 and / or the sense layer 144 before the base layer 112 and the sense layer 144 are aligned and connected.

[0167] Although the sensor electrode 126 can have minimal exposure to the fluid in the well, such as in the form of a nanowire, the inventors have proposed this example to optimize the performance and improve manufacturability of the nanopore sensor 102.

[0168] 2 and 3(a), the sensor electrode 126 is substantially planar and shaped to accommodate the passageway 114. In other words, the sensor electrode 126 is configured to allow unimpeded fluid communication between the analyte reservoir 106 and the outlet chamber 108, which can be achieved by (i) shaping the sensor electrode 126 to extend around the passageway 114 or well opening 142a, and / or (ii) forming a sensor opening 148 in the sensor electrode 126.

[0169] The footprint of the exposed portion of the sensor electrode 126 can be any shape. The well 142 of FIG. 2 and the cavity 146 of FIG. 3(a) can be cylindrical, such that the floor of the well 142 is circular, or the flat surface of the cavity 146 is curved. These configurations result in the exposed portion of the sensor electrode 126 being circular or disk-shaped. In the example shown, the sensor opening 148 is provided so that the footprint of the exposed portion is shaped like an annulus. The exposed area of ​​the sensor electrode 126 can be maximized, which can mean covering at least one face or surface of the well 142 and / or cavity 146.

[0170] Although the sensor electrode 126 and sensor aperture 148 are shown as circular, they may have other shapes. In some embodiments having a circular shape, the ratio of the radius of the exposed portion of the sensor electrode 126 to the radius of the sensor aperture 148 may be in the range of 100:1 to 10:1 or about 2:1. For non-circular shapes, the ratio of the square roots of the areas may have the same value.

[0171] Alternatively, the area of ​​the exposed portion of the sensor electrode 126 can be expressed in terms of the ratio between the area or footprint as viewed from the sensor opening 148, which can be about 1:1, or about 10:1, or about 100:1.

[0172] By way of example, the sensor electrode 126 may have a diameter (or maximum dimension) in the range of 10 μm to 50 μm, and the sensor opening 148 may have a diameter (or maximum dimension) in the range of 0.5 μm and up. The sensor opening 148 does not function as a sensor, and therefore its size has no upper limit within which it is desirable to minimize restriction of the remaining area of ​​the sensor electrode 126.

[0173] The sensor electrodes 126 may be formed from a suitable conductive material, such as platinum or gold.

[0174] While FIGS. 2 and 3(a) have sensor electrodes 126 with connections 128 to separate electronic circuitry 130, FIG. 3(b) illustrates that the structure 100 and array 104 can house an integrated circuit 150. The integrated circuit 150 can incorporate one or more of the functions of the electronic circuitry 130. In other words, various functions such as sensing, amplification, control, filtering, readout, etc. that can be implemented in separate electronic circuitry 130 can alternatively be implemented in the integrated circuit 150. The integrated circuit 150 can be formed on a separate layer or wafer and subsequently connected to the sense layer 144 having the sensor electrodes 126 thereon. However, the inventors contemplate that the integrated circuit 150 will be fabricated within the sense layer 144 along with the sensor electrodes 126. An integrated circuit 150 can be provided for each nanopore sensor 102.

[0175] In some approaches, after fabrication of the integrated circuit 150 and sensor electrodes 126 formed and the sense layer 144 exposed to one side, the sensing structure is inverted and bonded to a base layer in the same manner as was done in connection with FIG. 3( a). Connections 128 (not shown in FIG. 3( b)) connect the integrated circuit 150 to a connector 151 for transmitting signals or data generated by the integrated circuit 150 out of the structure. The connections 128 can be connected to the connector 151 as shown in FIG. 4( e), although other configurations are possible. With the sense layer 144 bonded to the base layer 112, the nanopore layer 110 can be formed thereon such that the sense layer 144 is sandwiched between the nanopore layer 110 and the base layer 112. As in FIG. 3( a) above, when bonded together, the passages in the sense layer 144 align with the channels in the base layer 112, and the wells 142 in the nanopore layer 142 form part of the passages 114.

[0176] In use, the electronic circuitry 130 and / or the integrated circuit 150 are configured to detect a resistance change in the nanopore 116 when an analyte, such as a polymer, passes through the nanopore 116, the resistance change being detected through a fluid within the nanopore sensor 102 (strictly speaking, as noted above, the measure of resistance is detected as a voltage across an effective voltage divider). In the array 104, the integrated circuit 150 of each nanopore sensor 102 may be communicatively addressable. In light of parasitic capacitance, noise from communications, and background noise, directly reading the detected resistance using an off-board processor may be difficult. To provide a better signal, i.e., a cleaner, noise-reduced signal, to the processor, the integrated circuit 150 may be configured to locally convert, modify, or otherwise process the signal derived from the detection of polynucleotides or other analytes passing through the nanopore 116. The integrated circuit 150 may be configured to at least one of amplify the signal, such as amplifying the voltage level of the signal, filter the signal, for example to remove noise, sample the signal, and digitize the signal using an analog-to-digital converter (ADC) implemented in the electronic circuit 130.

[0177] The integrated circuit 150 may be formed within the footprint of the nanopore sensor 102 in the array 104 of the structure 100 .

[0178] As an example, each nanopore sensor 102 in the array 104 may be contained within a pixel 101, which is the footprint of the nanopore sensor 102, as seen in FIG. 4(a), which can be considered to represent a schematic plan view of the nanopore sensor 102 shown in FIG. 3(b). As can be seen, each pixel 101 houses the nanopore sensor 102, a sensor electrode 126, and an integrated circuit 150. The sensor electrode 126 and the integrated circuit 150 may be arranged to suppress noise interference generated by the integrated circuit 150 from being detected by the sensor electrode 126. For example, the integrated circuit 150 may be separated from the nanopore sensor 102, as shown in FIG. 4(a). This separation may be achieved by configuring the integrated circuit 150 to be located outside of the pixel 101, as can be seen.

[0179] This separation can also simplify the manufacturing process. Alternatively, the integrated circuit 150 can be distanced from the sensor electrode 126 (i.e., the depth distance between the sensor electrode 126 and the integrated circuit 150, or the thickness of the structure 100, is increased to minimize noise interference. Note that the depth direction in FIG. 4(a) is the direction into or out of the page when viewed.

[0180] In the example shown, the pixel 101 is square with a side length of 20 μm, but in other examples may be in the range of 10 μm to 50 μm.

[0181] By way of example, the integrated circuit 150 occupies approximately three-quarters of the pixel 101, while the remaining quarter is occupied by the sensor electrode 126, which in the example shown has a diameter of 10 μm.

[0182] Other arrangements are envisioned. In some embodiments, the sensor electrode 126 may be larger than the example shown in FIG. 4( a), for example, covering substantially the entire area of ​​the nanopore sensor 102. In some embodiments, the sensor electrode 126 may have other shapes that cover more area, for example, a square or a rectangle. Depending on its shape, the sensor electrode 126 may have dimensions of up to 50 μm, in which case it may have dimensions of up to 250 μm. 2 The area of ​​the slit may be 0.05 mm.

[0183] For packaging efficiency, the pixels 101 may be tessellated, for example, the tessellation may be hexagonal.

[0184] Each nanopore sensor 102 has a passageway 114; however, during fabrication of the base layer 112, more channels 122 may be created in the base layer 112 than needed, depending on the fabrication method. Some fabrication methods, such as reactive ion etching, can etch a single channel 122 for each pixel 101. Other methods, such as light-assisted electrochemical etching, require simultaneously etching a dense array of channels 122 to maintain their shape. In this case, unused channels 122 in the base layer 112 are blocked during fabrication of the array 104, thereby providing only one channel 122 and one passageway 114 per pixel 101. The density of the channels 122 formed in the base layer 112 may vary. For comparison, FIG. 4(b) shows a cross-section of a nanopore sensor 102 with a lower density of blocked channels 122a than that shown in FIG. 4(a). As shown in Figure 4(b), the channel 122 may be blocked before the sense layer 144 is added to the base layer 112, or may be blocked by the substrate of the sense layer 144. Note that Figure 4(b) is shown with portions of two nanopore sensors 102, each with its own passageway 114, and that the nanopore layer 110 has not yet been added over the sense layer 144.

[0185] Figure 4(c) shows the pixels 101 of the nanopore sensors 102 of Figure 4(a) arranged in a 6x6 layout, providing an array 104 of 36 nanopore sensors 102, while Figure 4(d) has an 18x18 array with 324 nanopore sensors 102. The array size can be 1000x1000, providing 1,000,000 nanopore sensors 102. In this example, an array of 1 million nanopore sensors 102 of the type shown in Figure 4(a) would measure 4 cm 2 However, the nanopore sensor 102 with a small pixel 101 of 5 μm is 25 mm 2 The array 104 may have a footprint of up to 1 million nanopore sensors. The array size may be 100,000. The array 104 may include any number of nanopore sensors 102, from 1000 to 10,000,000 nanopore sensors 102.

[0186] FIG. 4(e) shows an array 104 having nanopore sensors 102 as described herein disposed in a structure 100 provided in a device 149 for receiving and analyzing polymeric analytes, such as nucleic acids. The device 149 may also be known as a sensor device or a measurement system. The array 104 may be a partial component of the device 149. The array 104 may be a disposable component and may be replaceable. Additionally or alternatively, the nanopore layer 110 of the array 104 may be a disposable component and may be replaceable. While some embodiments of the present invention are directed generally to the device 149, other embodiments of the present invention may also reside in the nanopore sensor 102 or a nanopore sensor 102 having an array of nanopore structures 104. The device 149 may include electronic circuitry 130 as described above.

[0187] In some embodiments, processing of the signal measured by nanopore sensor 102 may be performed by electronic circuitry 130. Integrated circuit 150 may perform pre-processing before further analysis by electronic circuitry 130 of device 149.

[0188] In some embodiments, the device 149 contains the structure 100 to separate and define an analyte reservoir 106 and an outlet chamber 108. Often referred to as cis and trans, respectively, analyte can flow from the analyte reservoir 106 to the outlet chamber 108. The array 104 has a plurality of nanopore sensors 102, each having a passageway 114 therethrough, to fluidly connect the analyte reservoir 106 and the outlet chamber 108. By way of example, actuation electrodes 132 in the analyte reservoir 106 and the outlet chamber 108 can impose a potential difference across the passageway 114 between the analyte reservoir 106 and the outlet chamber 108 to induce analyte to flow from the analyte reservoir 106 to the outlet chamber 108. The actuation electrodes 132 can be configured such that the potential difference is substantially the same across all of the nanopore sensors 102.

[0189] Additionally or alternatively, device 149 can be configured to use other techniques to direct the analyte from analyte reservoir 106 to exit chamber 108. As the analyte passes through nanopore 116, fluctuations in electrical potential caused by changes in ionic current flow are detected by sensor electrode 126.

[0190] The sensor electrode 126 can function as the base or gate of, or be directly connected to, a sensing transistor 153 (as shown in FIG. 5( a) and described further below), which can be, for example, a field effect transistor (FET) device. The sensing transistor 153 outputs a signal that can be processed by the integrated circuit 150 of each nanopore sensor 102, which can then be addressed in a row-column type manner. For example, the voltage at the drain of the sensing transistor 153 can depend on the potential sensed by the sensor electrode 126, and the drain voltage can be read in a row-column manner along with other drain voltages on other nanopore sensors 102 in the array 104. The processed signal can then be further analyzed outside the array 104 to determine one or more properties of the analyte.

[0191] While in the above example, each pixel 101 has its own integrated circuit 150, the integrated circuit 150 can be configured to service multiple nanopore sensors 102. In FIG. 4(f), four nanopore sensors 102 are shown as a sensor module 102a (which may be part of a larger array of nanopore sensors 102), with the integrated circuit 150 being common to four centrally located sensor electrodes 126 as shown. Other configurations are possible. In such modular configurations, information or data obtained from each individual nanopore sensor 102 is addressable for control and / or retrieval of information. While the above example has a dedicated integrated circuit 150 for each nanopore sensor 102, combining nanopore sensors 102 into a sensor module 102a can improve layout efficiency. Improved efficiency can be achieved, for example, because a common filter is used for each of the nanopores 116 in the sensor module 102a. This is possible if the integrated circuit 150 switches or multiplexes between the individual nanopore sensors 102 in sequence. By sharing functionality among nanopore sensors 102, the footprint of integrated circuit 150 can be reduced, or alternatively, can accommodate more functionality.

[0192] FIG. 5(a) is a schematic diagram of a direct connection of each nanopore sensor 102 in the array 104 to a sensor electrode 126. The cis electrode 132a can be connected to ground while a transfer voltage is applied to the trans electrode 132b. The resistance of the nanopore 116 and the resistance of the channel 122, configured to function as a fluidic resistor, complete a circuit between the cis electrode 132a and the trans electrode 132b through each passageway 114 of each nanopore sensor 102. In this way, the circuit behaves like a voltage divider with two resistors of similar value. The nanopore resistance and the resistance of the channel 122 or fluidic resistor are approximately the same so that an electrode positioned between them is optimally positioned to detect changes in the nanopore resistance caused by an analyte passing therethrough. A sensor electrode 126, as described above, is present in the region of each nanopore 116. The sensor electrode 126 can be located between the nanopore 116 and the channel 122. The effective impedance of nanopore 116 and channel 122 is much larger than the bulk fluidic resistance of analyte reservoir 106 and exit chamber 108. This means that Figure 5(a) can be used to model the circuit between cis electrode 132a and trans electrode 132b.

[0193] The circuit includes a sensing circuit 152 that measures the fluid potential at a sensor electrode 126 of the nanopore sensor 102 to take a measurement from the nanopore 116. The sensing circuit 152 may include a sensing transistor 153, which may be, for example, a field effect transistor (FET). In this case, the sensor electrode 126 may be connected to the base of the sensing transistor 153. The sensing circuit 152 may reside, at least in part, within an integrated circuit 150. Thus, the sensor electrode 126 may be connected to a sensor terminal 154 of the sensing circuit 152, as shown in FIG. 5( a).

[0194] Optionally, sensing circuit 152 may include control circuitry 155 that applies a signal to sensor electrode 126 to vary the potential difference across nanopore 116 imposed by drive electrode 132 in response to the control signal. Control circuitry 155 may include a control terminal 156, which may be, for example, a field effect transistor (FET). In this case, sensor electrode 126 may be connected to the drain of control terminal 156. Control circuitry 155 may reside, at least in part, within integrated circuit 150. Thus, sensor electrode 126 may be connected to control terminal 156 of control circuitry 155, as shown in FIG. 5( a), to apply a control signal.

[0195] Application of the control signal allows for varying the potential difference imposed across the individual nanopores 116 by varying the potential difference between the control connection of the control circuit 155 and the cis electrode 132a and / or the trans electrode 132b. The signal applied to the sensor electrode 126 can be a reverse voltage that induces a charged analyte, such as a species, to change direction of migration through the passageway 114.

[0196] In some cases, the applied voltage may be an AC voltage, although other voltage waveforms (eg, ramp, step, impulse, DC) may alternatively be applied.

[0197] 5(a) allows a common electrode to be configured for each of the analyte reservoirs 106 and the exit chambers 108, while each nanopore sensor 102 is operable to detect an interruption in ionic current flow across the passageway 114 by detecting a change in potential caused by a change in nanopore resistance. Additionally, the integrated circuit 150 allows each nanopore sensor 102 in the array 104 to be individually controlled, for example, to enable the sensor electrode 126 to detect an analyte passing through the nanopore 116 via connection with the sensing transistor 153, or to control the flow of charged analytes, such as species, through the passageway 114 of an individual nanopore sensor 102 in the array 104 by, for example, adjusting the voltage applied to the sensor electrode 126 using the control transistor 156. Controlling the flow of charged analytes, such as species, within the passageways 114 of individual nanopore sensors 102 in the array 104 allows analytes passing through or blocking the nanopores 116 to be repelled or expelled by voltages applied by the control transistors 156. This action, which can be described as "flicking" or "rejecting," is accomplished by using control voltages so that the flow of analytes through the passageways 114 from one side of the structure 100 is modified, i.e., stopped, reversed, or accelerated. Because each nanopore sensor 102 is individually addressable for control and sensing, a control voltage can be applied to each nanopore 116 individually. For clarity, application of a control signal to the sensor electrode 126 of each nanopore sensor 102 means that the voltage near the nanopore 116 at each pixel 101 can be controlled.

[0198] A control voltage can be applied to alter the movement of the analyte through the nanopore 116 in response to at least one of the following conditions: when the nanopore 116 is detected to be blocked, when the detected analyte is no longer of interest and should be expelled to allow another sample to be received and measured, and conditions including altering the rate at which the analyte is guided in and out of the nanopore 116.

[0199] Electronic sensors inevitably have capacitance, resistance, and inductance associated with the path the sensor signal travels. These are sometimes referred to as parasitics. These arise from the properties of the materials that make up the sensor, the sensor geometry, and the method by which it is feasible to fabricate the sensor. Without any kind of capacitance compensation, these parasitics (most commonly resistance and capacitance) interact to limit the bandwidth of the signal. In the simplest case, a resistor-capacitor circuit limits the bandwidth to 1 / (2πRC).

[0200] 5(b) is an alternative schematic diagram of FIG. 5(a), illustrating a resistor model 161 of the nanopore 116 and channel 122, and further including a compensation circuit 160 connected to a voltage divider. According to some embodiments, the compensation circuit 160 includes an in-line amplifier 168 with gain G connected to the output of the sensor electrode 126, which is subject to a parasitic input capacitance 162, also known as stray capacitance. The output of the in-line amplifier 168 has a feedback loop connected to its input, said feedback loop including a feedback amplifier 170 with gain H and a compensation capacitance C compensation It has a capacitor C representing

[0201] A parasitic input capacitance 162 is shown disposed in parallel with a resistor representing the channel 122, representing parasitic capacitance in at least one of the membrane 118 in which the nanopore 116 resides, the fluidic walls of the channel 122, the sensor electrode 126, and trace capacitance associated with a connector or wire bond. The sensor electrode 126 is effectively connected to the midpoint of a voltage divider between the nanopore 116 and the channel 122 and connected to the compensation circuit 160. The connection to the reverse or flick voltage is represented by a flick switch 164, such as a FET. An optional guard switch 166 is shown implemented between the sensor electrode 126 and the compensation circuit 160. This guard switch 166, which can be implemented using a FET, can function to isolate the compensation circuit 160 and / or any sensing circuitry connected thereto from the flick voltage applied via the flick switch 164.

[0202] Overall, the compensation circuit 160 reduces the effect of the total parasitic input capacitance 162 at the input to the sensing circuit 152. Parasitic capacitance exists in various elements of the nanopore sensor 102, which can be modeled as shown in FIG. 5(b). Without being bound to a particular theory, the total parasitic input capacitance 162C p can be considered as the sum of the parasitic capacitances as follows: C p =C membrane +C fluidic walls +C electrode +C trace

[0203] The rate at which the input capacitance charges is proportional to the current flowing through it. The resistor then limits the charging current to a finite value. The compensation circuit 160 functions to provide additional current to charge the input capacitance faster, thus increasing the bandwidth.

[0204] The compensation circuit 160 of Figure 5(b) includes an in-line amplifier 168 with gain G connected to the output of the sensor electrode 126, which is subject to the parasitic input capacitance 162. The output of the in-line amplifier 168 has a feedback loop to its input, which includes a feedback amplifier 170 with gain H and a compensation capacitance C compensation The compensation capacitor C172 represents

[0205] In some embodiments, the input voltage is amplified and fed back through a compensation capacitor 172 to provide additional current to charge the total parasitic input capacitance 162. The effective input capacitance of this circuit can be expressed as: C effective =C in -C compensation where: C compensation =C*(G*H-1)

[0206] The components of the compensation circuit 160 are the total parasitic input capacitance C p 5(b) 。 Compensation circuit 160 can compensate for a range of different parasitic input capacitance values ​​if the capacitance C, in-line gain G, or feedback gain H is made adjustable, and therefore feedback amplifier 170 is illustrated as variable in FIG. 5(b) . Gain G can be fixed, so that the output from compensation circuit 160 has a constant gain, and therefore either the compensation capacitor C 172 and / or the feedback gain H can be varied.

[0207] The front-end electronics may reside, at least in part, within the integrated circuit 150, figuratively represented in FIG. 5(c). Control circuitry 155 and / or compensation circuitry 160 may optionally be incorporated within the integrated circuit 150. The integrated circuit 150 or electronic circuitry 130 may be operable to affect the movement of the analyte within the nanopore 116, such as by flicking, applying a reverse voltage, and amplifying the signal from the nanopore sensor 102. The integrated circuit 150 or electronic circuitry 130 may further incorporate further processing of the signal, such as filtering, and, in the case of the integrated circuit 150, may include circuitry to store information locally to the sensor 102 for managed communication with an external processor.

[0208] Each nanopore sensor 102, as shown in Figures 2 and 3(a), may be addressable. Figure 5(c) represents the nanopore sensor 102 of Figure 3(b) incorporating an integrated circuit 150 within the pixel 101 shown in Figure 4(a), also addressable via row select and column buses. Figure 5(d) is an example of a row-column readout circuit 174 connected to each nanopore sensor 102 in an array 104, such as the array 104 shown in Figure 4(d), via the row select and column bus connections shown in Figure 5(c). Each nanopore sensor 102 is connected to a row decoder 176 and a column readout 178 via an analog-to-digital converter (ADC) 180. The row-column readout circuit 174 can be connected to the integrated circuit 150 of each nanopore sensor 102 or group of nanopore sensors 102, or it may be connected directly to the sensor electrodes 126 of each nanopore sensor 102 in the array 104.

[0209] The above example describes a sensor electrode 126 that is connectable to an integrated circuit 150 and has dual function options when a control voltage is applied (i.e., the sensor electrode 126 can be used to sense changes in ion flux as an analyte passes through the nanopore 116 and, under the control of the control circuit 155, to generate an electrical potential within the passageway 114 and a potential difference across the passageway 114 between the cis electrode 132a and / or the trans electrode 132b). In this case, the sensor electrode 126 is connected directly to a control terminal 156, which is a terminal of the integrated circuit 150, to generate an electrical potential within the passageway 114, as shown in FIG. 5(a) and described further below.

[0210] In some implementations, the sensing and control functions of each nanopore sensor 102 may be implemented by separate electrodes, for example, as follows: Figure 6(a) shows sensor electrode 126 and control electrode 182 arranged as an annulus, and Figures 6(b)-6(e) are cross-sectional schematic diagrams of nanopore sensors 102 having configurations in which control electrode 182 is provided in addition to sensor electrode 126. In this case, control electrode 182 is connected to control terminal 156 of control circuit 155 to generate a potential within passageway 114.

[0211] In the examples herein, the sensor electrode 126 is described as an annulus, as illustrated in FIG. 4(a). The sensor electrode 126 can also be implemented by an exposed wire. The sensor electrode 126 can be a nanowire, but can have a larger surface area, for example, occupying substantially the entire base of the well 142 or one side of the recess 146, as shown in FIG. 6(e). Similarly, the separate control electrode 182 can be a nanowire, but can have a larger surface area, as shown in FIG. 6(d).

[0212] From a manufacturability and cost perspective, a basic implementation of the control electrode 182 is shown in FIG. 6( a), where a ring footprint (suitable for the base of the well 142) is substantially maintained, with half of the footprint formed by the sensor electrode 126 and the other half physically disconnected or decoupled from the sensor side to form the control electrode 182. There are no wires or solid connections between the sensor electrode 126 and the control electrode 182. The sensor electrode 126 and the control electrode 182 are shown as having two equally sized semicircular shapes occupying the footprint. The electrodes can be different sizes; for example, the control electrode 182 can have a larger surface area than the sensor electrode 126 to increase conductivity with respect to the fluid in the passageway 114.

[0213] Having separate sensor electrode 126 and control electrode 182 can simplify integrated circuit 150 because the separation provides an additional degree of isolation, even though they are still connected via the fluid in passageway 114. However, it may be possible to avoid the need for an isolation switch to protect, for example, compensation circuit 160, which may form part of sensing circuit 152, from voltages applied by control circuit 155. Sensor electrode 126 and control electrode 182 can be shaped, sized, and configured to be optimized for their purposes.

[0214] Figure 6(b) shows how the sensor electrode 126 of Figure 3(b) can be divided into separate sensor electrode 126 and control electrode 182. In this example, the sensor electrode 126 and control electrode 182 extend in the same plane. In an alternative configuration shown in Figure 6(c), the sensor electrode 126 resides in the cavity 146 and extends in a plane extending parallel to the cis surface 134 and trans surface 138, while the control electrode 182 extends within the channel 122 and extends perpendicular to the cis surface 134 and trans surface 138. In Figure 6(c), the sensor electrode 126 is shaped like an annulus, and the control electrode 182 is shaped like a cylinder. In yet another alternative, as shown in Figure 6(d), the sensor electrode 126 resides within the cavity 146 and extends in a plane extending parallel to the cis surface 134 and the trans surface 138, while the control electrode 182 extends within the channel 122 and the cavity 146 and thus, as observed, extends in vertical and horizontal planes. Figure 6(e), similar to Figure 6(b), shows the sensor electrode 126 and the control electrode 182 formed at the base of the well 142, which may allow for easier fabrication.

[0215] As noted above, electronic sensors necessarily have capacitance, resistance, and inductance associated with the path the sensor signal travels. This may be referred to as parasitics, including parasitic capacitance. In addition to, or instead of, the compensation circuit 160 described above, the array 104 and nanopore sensors 102 therein may be fabricated incorporating guard conductors 184, as shown in Figures 7(b)-7(d), while Figure 7(a) shows a first schematic circuit 201 without guard conductors 184 and a second schematic circuit 202 with guard conductors 184 to illustrate how the guard conductors 184 may be configured.

[0216] In the first schematic circuit 201 on the left side of FIG. 7(a), the parasitic capacitance C parasiticis shown between two conductive elements 203, 204 of the nanopore sensor 102, which are typically conductors such as the sensor electrode 126 and the conductive substrate of the base layer 112. The first conductive element 203 (e.g., the sensor electrode 126) is connected to a voltage V sensor , and the second conductive element 204 (e.g., a conductive substrate) can carry a different voltage V substrate can be carried.

[0217] The guard is shown implemented in the second schematic circuit 202 on the right, where a third conductive element, the guard conductor 184, has two parasitic capacitances C between the first conductive element 203 and the second conductive element 204 that carry the signal. par1 , C par2 In this second schematic circuit 202, the parasitic capacitance occurs between (i) the signal-carrying first conductive element 203 and the guard conductor 184, i.e., C par1 and (ii) a current generated between the guard conductor 184 and the second conductive element 204, i.e., C par2 A buffer 205 (which may be an amplifier) ​​is connected between the first conductive element 203 and the guard conductor 184 to apply a buffered version of the input signal to the guard conductor 184. As a result, a parasitic capacitance C par1 There is no voltage difference across the

[0218] For a capacitor, the current is given by I=C(dV / dt).

[0219] In the schematic on the right, V guard =V sensor , therefore dV / dt=0.

[0220] Capacitor C par1Since no current flows through the buffer 205, the effective capacitance is zero. The capacitance between the guard conductor 184 and the substrate conductor still must be charged, but the buffer 205 can source much more current than the high impedance sensor input, so the capacitance charges much faster.

[0221] These conditions are V guard V sensor This depends on the performance of the buffer 205 having sufficient bandwidth to allow the capacitance to be zeroed. A precise buffer 205 with a bandwidth of several MHz can be implemented.

[0222] FIG. 7(b) is similar to FIG. 2(b) and, for comparison, shows the guard conductor 184 extending between the oxide layer 192 along the length of the channel 122, as viewed vertically, and continuing horizontally along the top of the base layer 112 below the sensor electrode 126. Notably, both the sensor electrode 126 and the guard conductor 184 are connected to separate electronic circuits 130. In this configuration, the guard conductor 184 suppresses current flow in the parasitic capacitance between the sensor electrode 126 and the substrate of the base layer 112. The conductive guard may include, at least in part, the guard conductor 184 and an insulating layer, such as the oxide layer 192, that insulates the guard conductor 184 from the guarded conductor or from the conductor it guards. The insulating layer is not a part of the guard conductor 184, but rather functions to isolate the guard conductor 184 from the surrounding conductors. Thus, the insulating layer may be a non-conductive component of the structure 100. The guard conductor 184 may be a conductor inserted in the middle of the parasitic capacitance, dividing it into two halves. This is possible because the capacitor is inherently an insulator and therefore the guard conductor 184 is disposed within an existing insulating layer.

[0223] A conductive guard including an insulating layer extends across at least a portion of the nanopore layer 110 to isolate the nanopore layer 110 from the analyte in the analyte reservoir 106, protecting the solution below the nanopore 116 from the solution above, as shown in FIG. 7(c), and extends between at least a portion of the nanopore layer 110 and the sense layer 144 to isolate the sensor electrode 126 and the integrated circuit 150 from the solution in the nanopore 116, as also shown in FIG. 7(c). , as also shown in FIG. 7(c), extending between the base layer 112 and the sense layer 144 to at least partially isolate the sensor electrode 126 and the integrated circuit 150 from the base layer 112; and multiple conductive guards, as shown in FIG. 7(d), where a first conductive guard extends between the wall of the channel 122 and the base layer 112 and a second conductive guard extends between the sense layer 144 and the base layer 112.

[0224] In light of the teachings herein, one skilled in the art will appreciate that one or a combination of the guard arrangements taught herein can be implemented. It will also be appreciated that guard conductors 184 can be provided within an array 104 of nanopore structures, such as the array 104 of FIG. 4(c).

[0225] Note that the guard-based capacitance compensation techniques shown in FIGS. 7(a)-7(d) have the advantage that they generally do not appreciably increase the noise level of the signal. However, such techniques cannot compensate for membrane capacitance when a potential difference across the membrane 118 is used to drive the analyte being studied through the nanopore 116, although it may be possible to drive the analyte by other means, such as pressure. On the other hand, the compensation circuit 160 can compensate for the entire input capacitance, but at the cost of adding noise. The noise gain of the compensation capacitor 172 increases with frequency. Therefore, the noise of the input signal is scaled by this feedback gain "G" and added to the overall noise. This becomes significant at higher frequencies or when compensating for larger input capacitances. Therefore, the guard conductors 184 shown in FIGS. 7(b)-7(d) can be implemented in the array 104 in any combination and / or in combination with the compensation circuit 160.

[0226] The nanopore sensors 102 can be fabricated using several different techniques, and their function is taught by way of example with reference to FIG. 2 , which illustrates other sensors taught in this application. While only one of the nanopore sensors 102 of the array 104 is shown in FIG. 2 , fabrication of the array 104 can be understood from the teachings herein. The base layer 112 is formed from a standard silicon (Si) wafer having channels 122 formed therein to pass from one side of the base layer 112 to the other. Only one channel 122 formed through the Si wafer, extending substantially perpendicular to the surface of the wafer, is shown in FIG. 2 . In practice, the array 104 has channels 122 formed across the wafer using techniques such as photolithography or deep reactive ion etching (DRIE), or a combination thereof. At least one channel 122 is formed for each nanopore sensor 102. If necessary, techniques such as thermal oxidation can be used to adjust the diameter of the channels 122 and calibrate the aspect ratio. The Si wafer and channels can be embedded in an oxide layer, which can be formed on a silicon wafer, for example.

[0227] The example of Figure 2 schematically illustrates a portion of a structure 100 having an array 104 of nanopore sensors 102 (only one of which is shown) configured to separate an analyte reservoir 106 and an outlet chamber 108 having a driving electrode 132 therein. All nanopore sensors 102 herein can be arranged within the structure 100 as shown in Figure 4(e). The nanopore 116 is located within a passage 114 between the analyte reservoir 106 and the outlet chamber 108, which is filled with fluid. The passage 114 is filled with fluid such that the analyte reservoir 106 and the outlet chamber 108 are fluidly connected. For clarity, the nanopore 116 is in the fluid communication path between the analyte reservoir 106 and the outlet chamber 108.

[0228] Figures 8 and 9 show two further examples of devices 149 including structure 100. In each case, structure 100 takes the form shown in either Figure 3(a) or 3(b), including a nanopore layer 110, a sense layer 144, and a base layer 112, as described in detail above (although in each case this could be replaced by structure 100 taking the form shown in Figure 2).

[0229] In each of the examples of Figures 8 and 9, structure 100 separates analyte reservoir 106 and outlet chamber 108 and is connected to printed circuit board 210, but in different configurations as follows.

[0230] In the example of Figure 8, the analyte reservoir 106 and the outlet chamber 108 are each formed by respective gaskets 216, 218 that seal the nanopore layer 110 and the base layer 112, respectively. The analyte reservoir 106 and the outlet chamber 108 may be open, as shown in Figure 8, or may be closed, for example, by respective members extending across the gaskets 216, 218.

[0231] In the example of Figure 8, printed circuit board 210 is attached to base layer 112 by mechanical bond 212 (adhesive) on the opposite side from nanopore layer 110. Printed circuit board 210 is therefore positioned outside of outlet chamber 108, as shown in Figure 8. Sense layer 144 is connected to printed circuit board 210 by wire bonds 214, or any other suitable electrical connection. Nanopore layer 110 has a smaller area than sense layer 144 to provide space for wire bonds 214.

[0232] 9, the printed circuit board 210 is attached to the sense layer 144 by solder bump connections 222 (e.g., adhesive) on the same side as the nanopore layer 110. The nanopore layer 110 therefore has a smaller area than the sense layer 144 to provide space for the solder bump connections 222. The solder bump connections 222 provide both a mechanical and an electrical connection between the printed circuit board 210 and the sense layer 144.

[0233] 9, the analyte reservoir 106 and the outlet chamber 108 are each formed within a respective flow cell 224, 226, which may be made from any suitable material, such as, for example, plastic. The flow cells 224, 226 allow fluid to flow into and out of the analyte reservoir 106 and the outlet chamber 108.

[0234] A flow cell 224 forming the analyte reservoir 106 is sealed to the printed circuit board 210 around the analyte reservoir 106 by a gasket 228, and the printed circuit board 210 is sealed to the edge of the nanopore layer 110 around the analyte reservoir 106 by a sealant 230.

[0235] The flow cell 224 that forms the outlet chamber 108 is sealed to the base layer 112 around the outlet chamber 108 by a gasket 232 .

[0236] The examples of Figures 8 and 9 can be modified in various ways and by any suitable means, for example, to provide a seal in other locations (eg, around the outer edge of the base layer 112).

[0237] The electrical model of the nanopore sensor 102 is described above. More generally, a voltage source, not shown in FIG. 2, applies a potential difference between the driving electrode 132 configured in the analyte reservoir 106 and the exit chamber 108. The driving electrode 132 imposes a potential across the passageway 114, which includes the nanopore 116 and the channel 122. The nanopore resistance and the channel resistance are significantly higher than the overall fluidic resistance of the analyte reservoir 106 and the exit chamber 108; therefore, the nanopore 116 and the channel 122 are key components in an equivalent electrical circuit. As shown in FIG. 2, the sensor electrode 126 is positioned between the nanopore 116 and the channel 122 so as to be able to sense the fluid potential at an electrical transduction element within the passageway 114. In other words, the sensor electrode 126 can sense a signal indicative of local potential fluctuations in the passageway 114. While the configuration of FIG. 2 is exemplary, the sensor electrode 126 can be located in the analyte reservoir 106 or the exit chamber 108. The sensor electrode 126 can function as the base or gate of a transistor device to measure the potential of the fluid at the location of the sensor electrode 126 when the fluid is provided to the passageway 114. The sensor electrode 126 can detect a change in voltage as an object of a species, such as a strand of DNA, moves through the nanopore 116.

[0238] Embodiments herein have described a device 149 having a single analyte reservoir 106 separated from a single outlet chamber 108 by structure 100. In light of the teachings herein, alternative arrangements may be implemented, including a device 149 having (i) two or more analyte reservoirs 106 separated from a common outlet chamber 108 by structure 100, (ii) a common analyte reservoir 106 separated from two or more outlet chambers 108 by structure 100, or (iii) two or more analyte reservoirs 106 separated from two or more respective outlet chambers 108 by structure 100.

[0239] The nanopore layer 110 can be separately formed with an array of wells 142, which can be formed in several ways, one of which is by lithographic patterning of a polymer layer. The wells 142 in the nanopore layer 110 are then aligned with the channels 122 in the base layer 112 so that each nanopore sensor 102 has a passage 114 defined by the wells 142 and the channels 122. The wells 142 shown in FIG. 2 are substantially larger than the nanopores 116 disposed in the membrane 118. The nanopores 116 in FIG. 2 are biological nanopores in the membrane 118, such as an amphiphilic membrane. Alternatively, each nanopore 116 can be a solid-state nanopore located in a solid membrane. The solid membrane itself can be the nanopore layer 110. Alternatively, the nanopores 116 can be biological nanopores disposed in a solid membrane. In light of the dimensions of nanopore 116 relative to the width of larger diameter channel 122, well 142 can be said to be formed below nanopore 116. Nanopore 116 therefore defines a portion of passageway 114 in each of the alternative nanopore configurations.

[0240] Any membrane 118 can be used in accordance with the various embodiments described herein. Suitable membranes 118 are well known in the art. The membrane 118 can be an amphiphilic layer or a solid layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. The amphiphilic molecules can be synthetic or natural. Non-natural amphiphiles and amphiphiles that form monolayers are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). The copolymers can be triblock, tetrablock, or pentablock copolymers. The membrane 118 can be a triblock or diblock copolymer membrane.

[0241] Membranes 118 formed from block copolymers hold several advantages over biological lipid membranes: Because triblock copolymers are synthetic, they can be carefully controlled to provide the correct chain length and properties needed to form membranes and interact with pores and other proteins.

[0242] Block copolymers may also be constructed from subunits that are not classified as lipid submaterials; for example, hydrophobic polymers may be made from siloxanes or other non-hydrocarbon monomers. The hydrophilic subsections of the block copolymer may also possess low protein binding properties, allowing for the creation of membranes 118 that are highly resistant when exposed to live biological samples. The headgroup units may also be derived from non-classified lipid headgroups.

[0243] Triblock copolymer membranes also have increased mechanical and environmental stability compared to biological lipid membranes, e.g., much higher operating temperature or pH ranges. The synthetic nature of block copolymers provides a platform for customizing polymer-based membranes for a wide range of applications.

[0244] Membrane 118 may be one of the membranes disclosed in WO2014 / 064443 or WO2014 / 064444, which are incorporated herein by reference in their entirety. These documents also disclose suitable polymers.

[0245] The amphiphilic molecule may be chemically modified or functionalized to facilitate binding of the polynucleotide.

[0246] The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer can be curved. The amphiphilic layer can be supported. The amphiphilic layer can be concave. The amphiphilic layer can be suspended from raised pillars so that the peripheral regions of the amphiphilic layer (attached to the pillars) are higher than the amphiphilic layer regions. This allows the microparticles to move, migrate, slide, or roll along the membrane as described above.

[0247] The membrane 118 may be a lipid bilayer. Suitable lipid bilayers are disclosed in WO2008 / 102121, WO2009 / 077734, and WO2006 / 100484.

[0248] Methods for forming lipid bilayers are known in the art. Lipid bilayers are generally formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA, 1972; 69:3561-3566), in which a lipid monolayer is supported on either side of an opening perpendicular to an aqueous solution / air interface.

[0249] The solid layer can be formed from both organic and inorganic materials, including, but not limited to, fine electronic materials, insulating materials such as Si3N4, Al2O3, and SiO2, organic and inorganic polymers such as polyamides, plastics such as Teflon® or elastomers such as two-component addition-cured silicone rubber, and glass. The solid layer may also be formed from graphene. A suitable graphene layer is disclosed in WO2009 / 035647. Yusko et al., Nature Nanotechnology, 2011;6:253-260, and U.S. Patent Application Publication No. 2013 / 0048499, describe the delivery of proteins to membrane-spanning pores in a solid layer without the use of microparticles.

[0250] Any transmembrane pore may be used. The nanopore 116 may be biological or artificial. Suitable nanopores 116 include, but are not limited to, protein pores, polynucleotide pores, and solid pores. The nanopore 116 may also be a DNA origami pore (Langecker et al., Science, 2012;338:932-936).

[0251] The transmembrane pore may be a transmembrane protein pore. A transmembrane protein pore is a polypeptide or assembly of polypeptides that allows hydrated ions, such as byproducts of polynucleotide processing by a polymerase, to flow from one side of a membrane 118 to the other side of the membrane 118. In one embodiment, the transmembrane protein pore can form a nanopore 116 that allows hydrated ions, driven by an applied potential, to flow from one side of the membrane 118 to the other. The transmembrane protein pore allows polynucleotides to flow from one side of a membrane 118, such as a triblock copolymer membrane, to the other. The transmembrane protein pore allows polynucleotides, such as DNA or RNA, to translocate through the nanopore 116.

[0252] The transmembrane protein pore can be a monomer or an oligomer. The pore can be composed of several repeating subunits, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 subunits. The pore can be a hexameric pore, a heptameric pore, an octameric pore, or a nonameric pore. The pore can be a homo-oligomer or a hetero-oligomer.

[0253] A transmembrane protein pore typically contains a barrel or channel through which ions can flow. The subunits of the pore typically surround a central axis and provide strands into a transmembrane beta barrel or channel or a transmembrane alpha-helical bundle or channel. The barrel or channel of a transmembrane protein pore usually contains amino acids that facilitate interaction with nucleotides, polynucleotides, or nucleic acids. These amino acids are located near the constriction of the barrel or channel. A transmembrane protein pore typically contains 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 interaction between the pore and nucleotides, polynucleotides, or nucleic acids.

[0254] The transmembrane protein pore for use in accordance with the present invention may be derived from a β-barrel pore or an α-helical bundle pore. The transmembrane pore may be derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1, and the hemolytic protein fragacetoxin C (FraC). The transmembrane protein pore may be derived from CsgG. A suitable pore derived from CsgG is disclosed in WO2016 / 034591. The transmembrane pore may be derived from lysenin. A suitable pore derived from lysenin is disclosed in WO2013 / 153359.

[0255] The analyte (including, for example, a protein, peptide, molecule, polypeptide, or polynucleotide) may be present in a sample. The analyte may be any suitable sample. The analyte may be a biological sample. Any embodiment of the methods described herein may be performed in vitro on a sample obtained or extracted from any organism or microorganism. The organism or microorganism is typically an archaea, prokaryote, or eukaryote, and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera, and protista. In some embodiments, the methods of various aspects described herein may be performed in vitro on a sample obtained or extracted from any virus.

[0256] The analyte may be a fluid sample. The analyte may include a bodily fluid. The bodily fluid may be obtained from a human or an animal. The human or animal may have, be suspected of having, or be at risk for a disease. The analyte may be urine, lymph, saliva, mucus, semen, or amniotic fluid, but may also be whole blood, plasma, or serum. Typically, the sample is from a human, but may alternatively be from another mammal, for example, from commercial livestock such as a horse, cow, sheep, or pig, or alternatively, from a pet animal such as a cat or dog.

[0257] Alternatively, the analyte may be of plant origin.

[0258] The analyte may be a non-biological sample. The non-biological sample may be a fluid sample. An ionic salt, such as potassium chloride, may be added to the sample to achieve ionic flow through the nanopore.

[0259] The polynucleotide may be single-stranded or double-stranded. At least a portion of the polynucleotide may be double-stranded.

[0260] A polynucleotide can be a nucleic acid such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A polynucleotide can contain one strand of RNA hybridized to one strand of DNA. A polynucleotide can 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. A polynucleotide can be of any length.

[0261] Any number of polynucleotides can be investigated. For example, the method can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. When two or more polynucleotides are characterized, they can be different polynucleotides or two instances of the same polynucleotide.

[0262] The polynucleotide may be natural or artificial.

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

[0264] For (iii), the sequence of the polynucleotide can be determined as described above. Suitable sequencing methods, particularly those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19): 7702-7, Lieberman KR et al., J Am Chem Soc. 2010; 132(50): 17961-72, and International Application WO2000 / 28312.

[0265] Secondary structure can be measured in a variety of ways. For example, if the method involves electrical measurements, the secondary structure can be measured using changes in residence time or changes in ionic current passing through the pore. This makes it possible to distinguish between regions of single-stranded and double-stranded polynucleotides.

[0266] The presence or absence of any modification can be measured.The method includes determining whether the polynucleotide is modified by methylation, oxidation, damage, one or more proteins, or one or more labels, tags, or spacers.Specific modifications will result in specific interactions with the pore, which can be measured using the following method.

[0267] In some embodiments of the various aspects described herein, the method can include further characterizing the target polynucleotide. When the target polynucleotide contacts the pore, one or more measurements indicative of one or more properties of the target polynucleotide are taken as the polynucleotide translocates relative to the pore.

[0268] The method can include determining whether the polynucleotide is modified. The presence or absence of any modification can be measured. The method can include determining whether the polynucleotide is modified by methylation, oxidation, damage, one or more proteins, or one or more labels, tags, or spacers.

[0269] Also provided is a kit for characterizing a target polynucleotide. The kit comprises a pore as disclosed herein and a membrane component. The membrane can be formed from the components. The pore can be present within the membrane. The kit can include any of the components of the membrane disclosed above, such as an amphiphilic layer or a triblock copolymer membrane.

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

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

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

[0273] The apparatus may be configured to perform the methods as disclosed herein.

[0274] The apparatus can include a sensor device capable of supporting a plurality of pores and membranes and operable to perform characterization of an analyte using the pores and membranes, and at least one port for delivering material to perform the characterization.

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

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

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

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

[0279] The features of the above description and drawings are interchangeable and compatible in light of the teachings of this specification. The present invention has been described above purely by way of example, and modifications can be made within the spirit and scope of the present invention, ranging from equivalents of the described features to combinations of one or more features described herein. The present invention also consists of any individual feature described or implied herein. [Explanation of symbols]

[0280] 2. Sensor Devices 4. Solid-state nanopores 6 Analytes 8 Main Unit 10. Cis Reservoir 12 Fluid passage 14 Trans Reservoir 16 sensors 18 electrodes 100 Structure 101 pixels 102 Nanopore structure 102a Sensor Module 104 Nanopore Structure Array 106 Analyte reservoir 108 Exit Chamber 110 Nanoporous layer 112 Base layer 114 Passage 116 Nanopores 118 Membrane 120 first end 122 channels 122a Blocked Channel 124 Second End 126 Sensor Electrode 128 connections 130 Electronic circuits 132 Drive electrode 132a cis electrode 132b Transformer electrode 134 cis surface 136 cis face 138 Transformer Surface 140 Trans Surface 142 wells 142a Well opening 144 Sensation Layer 146 Cavity 148 Sensor opening 149 devices 150 Integrated Circuits 151 Connector 152 Detection circuit 153 Detector transistor 154 Sensor terminal 155 Control circuit 156 Control terminal 160 Compensation circuit 161 Resistor Model 162 Capacitor 164 Flick Switch 166 Guard Switch 168 In-line Amplifier 170 Feedback Amplifier 172 Compensation Capacitor 174 Row-column readout circuit 176 Line Decoder 178 column readout 180 ADC 182 Control electrode 184 Guard Conductor 192 oxide layer 201 First schematic circuit 202 Second schematic circuit 203 First Conductive Element 204 second conductive element 205 buffers 210 Printed Circuit Board 212 Mechanical bonding 214 Wire Bond 216 Gasket 218 Gasket 222 Solder Bump Connection 224 flow cell 226 Flow Cell 228 Gasket 230 sealant 232 Gasket

Claims

1. A device for sensing an analyte, the device having a plurality of nanopore structures configured within a structure disposed to separate an analyte reservoir and an exit chamber, each nanopore structure providing a passage for fluid communication through the structure between the analyte reservoir and the exit chamber; Each nanopore structure is an electrical conversion element; an electronic circuit configured to detect and optionally amplify a signal from the electrical transduction element; each of the nanopore structures configured to one or more of store, transmit, process, and communicate at least a portion of the signal to a connectable processor; A device wherein each of said nanopore structures has a control terminal, each control terminal associated with a respective nanopore for applying a control signal to vary the potential difference across the nanopore.

2. The device of claim 1 , wherein the structure is configured to separate the analyte reservoir for receiving an analyte and the outlet chamber for collecting the analyte.

3. The device of claim 1 , wherein each of the nanopore structures within the structure further comprises a compensation circuit.

4. The device of claim 3 , wherein the compensation circuit comprises a variable gain amplifier and / or a variable capacitor in a feedback loop of the compensation circuit.

5. A device according to any preceding claim, wherein the control terminal is switchably connected to a power source to vary a configurable voltage level imposed across the nanopore.

6. The device of any one of claims 1 to 5, wherein each nanopore structure within the structure is organized into a pixel.

7. The device of claim 6 , wherein the pixels form a mosaic array of nanopore structures.

8. 1. A method of forming a device having a nanopore structure for sensing an analyte, comprising: forming nanopore structures within the structure and disposing the structures to separate an analyte reservoir and an exit chamber of the device, such that each nanopore structure provides a passage for fluid connection through the structure between the analyte reservoir and the exit chamber; Within each nanopore structure, an electrical conversion element; and an electronic circuit configured to measure a signal from the electrical transduction element; each of the nanopore structures is configured to store, transmit, process, and / or communicate at least a portion of the signal being measured or information derived therefrom to a connectable processor; The method wherein each of said nanopore structures has a control terminal, each control terminal associated with a respective nanopore for applying a control signal to vary the potential difference across the nanopore.

9. 10. The method of claim 8, further comprising configuring an analyte reservoir for receiving an analyte and an outlet chamber for collecting the analyte, and configuring a nanopore layer to separate the analyte reservoir and the outlet chamber.

10. 10. The method of claim 9, further comprising configuring actuation electrodes connected within the analyte reservoir and the exit chamber, respectively, to impose a potential difference across the passage of the nanopore structure.

11. The method of claim 10 , wherein the imposed potential difference is common across a plurality of the nanopore structures.

12. 12. The method of claim 10 or 11, further comprising configuring the electronic circuit with a switchable connection to apply a signal to a control terminal of each of the electrical transduction elements to vary the potential imposed by the drive electrode across each respective nanopore structure.

13. 13. The method of any one of claims 10 to 12, further comprising forming a control electrode within the passageway of each nanopore sensor, the control electrode selectively connectable to a signal for varying the potential imposed by the drive electrode across each respective nanopore structure.

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