Nanopore sensing devices, components, and operating methods

Abstract

A nanopore sensing device has a structure (100) arranged to separate an analyte reservoir (106) from an outlet chamber (108). The structure (100) includes an array of nanopore structures (104), each nanopore structure (104) including a passageway (114) for fluidly connecting the analyte reservoir (106) and the outlet chamber (108). A control terminal (156) is arranged to apply a control signal to change the potential difference across the nanopore structure (104). An electronic circuit (152) is configured to detect a signal from an electrical transducer element (126) at each nanopore structure (104).

Description

[0001] The present invention generally relates to an apparatus for nanopore sensing having an array of nanopore structures configurable as a nanopore sensor and methods for operating a nanopore sensor or fabricating an array of nanopore structures.

[0002] Nanopore sensors have been developed for sensing a wide range of species, including single molecules such as polymer molecules. A known nanopore sensor device is the MinION manufactured and sold by Oxford Nanopore Technologies Ltd. TM Nanopore-based sensing employs the measurement of ionic currents passing through biological nanopores located in highly resistive amphiphilic membranes. TM A nanopore sensor array is provided. When a molecule (such as a polymer analyte, e.g., DNA) is caused to displace the nanopore, the fluctuations in the ionic current can be measured to determine the sequence of the DNA strand. A nanopore device for detecting analytes other than polynucleotides (e.g., proteins) is also known from WO 2013 / 123379.

[0003] Biological nanopore devices (such as MinION TM ) are solid-state nanopore devices. Figure 1 A portion of a single sensor device 2 having a solid-state nanopore 4 as disclosed in WO2016 / 127007 (which is hereby incorporated by reference in its entirety) is shown, wherein: an analyte 6 passes from a cis-reservoir 10 through a body 8, through the solid-state nanopore 4, and into a fluid passage 12; a signal is read by a sensor 16 positioned proximate to the solid-state nanopore 4. Electrodes 18 are provided in the cis-reservoir 10 and the trans-reservoir 14 to induce the analyte 6 to pass through the solid-state nanopore 4.

[0004] The performance of solid-state nanopore sensors is limited by the tolerances of the sensing components, manufacturing techniques, and variations in the formation of the nanopore or assembly of the sensor. These and other factors compromise 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 a nanopore sensor array having a plurality of nanopore sensors.

[0006] The present inventors have sought to improve upon known nanopore sensing devices by providing the ability to control analyte movement in some aspects while also improving measurement accuracy by mitigating factors that hinder measurement, such as noise caused by parasitic and contaminated sensing components. Furthermore, the improved devices allow nanopore structures and nanopore sensors implemented thereby to be efficiently formed into large arrays without inhibiting array control or performance.

[0007] In a first aspect, the present invention provides an apparatus for performing nanopore sensing, the apparatus having: a structure arranged to separate an analyte reservoir from an outlet chamber, the structure comprising an array of nanopore structures, each nanopore structure comprising a passageway through the structure for enabling fluid connection between the analyte reservoir and the outlet chamber;

[0008] a drive electrode connected to the analyte reservoir and the outlet chamber, respectively, to impose a potential difference across the pathway;

[0009] electrical transduction elements, each element connected to or exposed to the passage of a corresponding nanopore structure to measure a fluid potential at the electrical transduction element in the nanopore structure; and

[0010] Control terminals, each connected to a corresponding nanopore structure, for applying a control signal to change the potential difference across the nanopore structure or to change the potential within the pathway.

[0011] The structure may be a support structure. The nanopore structure may be arranged in and / or on a corresponding array of passages. Each nanopore structure may have an orifice forming a portion of the passage. Each nanopore structure in the array of nanopore structures has a corresponding passage. The control terminal may be connected to a corresponding passage in the structure to apply a control signal to change the fluid potential distribution around the corresponding nanopore structure. When a fluid is provided so that a fluid connection exists between the drive electrode and the nanopore structure, the control signal applied to the nanopore structure may change the potential difference across the nanopore structure relative to the drive electrode. The control terminal may be connected to the electric transducer element. The control terminal may be switchably connected to the electric transducer element.

[0012] The nanopore structures of the array may have nanopores, be capable of supporting nanopores, or be capable of supporting a membrane having nanopores.

[0013] When operating as a nanopore sensing device, the device comprises an array of nanopores.

[0014] When a fluid is provided, a fluid potential can be measured at the electrical transduction element.When a fluid is provided, an electrical distribution of the fluid around the nanopore structure can be changed.

[0015] In operation, fluid resides in the analyte reservoir, the outlet chamber and the passageway of the device, wherein the reservoir and the chamber are fluidically connected. The fluids in the reservoir, the chamber and the passageway of the nanopore structure may be different fluids.

[0016] The nanopore structure may include a pore with a width of nanometer size. The pore may be a through hole in a solid support, such as a solid-state nanopore.

[0017] Alternatively, in an embodiment, the nanopore structure can be a structure capable of supporting a nanopore to provide a nanometer-sized passage. In this embodiment, the nanopore structure can include an orifice of micrometer or nanometer size. Exemplary nanopore structures that can be used to support nanopores are disclosed in WO2014064443, which is hereby incorporated by reference in its entirety. Examples of nanopores that can be supported by a nanopore structure are biological nanopores, such as protein nanopores. The nanopore can be provided in a membrane (such as an amphiphilic membrane). The membrane can be supported by the nanopore structure.

[0018] When used for nanopore sensing, the device may comprise an array of nanopores.

[0019] The analyte reservoir can be used to receive analytes for sensing by the nanopore array.The outlet chamber can be used to receive analytes that pass through the nanopore array.

[0020] The nanopore (where present) separates the cis side from the trans side of the device.The analyte reservoir can be considered the cis side of the device, and the analyte outlet chamber can be considered part of the trans side.

[0021] The device may be provided with or without fluid.The fluids in the analyte reservoir, the outlet chamber and the passageway of the nanopore structure may be different fluids.

[0022] In another aspect, the present invention provides a structure comprising an array of nanopore structures, each nanopore structure comprising a passageway for fluidic connection through the structure. Each nanopore structure has an electrical transduction element, each element connected to or exposed to the passageway of the corresponding nanopore structure, for measuring the potential of a fluid at the electrical transduction element in the nanopore structure. Each nanopore structure also has a control terminal, each control terminal connected to the corresponding nanopore structure for applying a control signal to change the potential distribution of the fluid in the passageway or surrounding the corresponding nanopore structure.

[0023] The structure may be a support structure. The nanopore structure may be arranged in and / or on a corresponding array of passages. Each nanopore structure may have an orifice forming a portion of the passage. Each nanopore structure in the array of nanopore structures may have a corresponding passage. The control terminal may be connected to a corresponding passage in the structure to apply a control signal to change the potential distribution of the fluid around the corresponding nanopore structure. Each orifice in the array may be associated with a corresponding electrical transducer element and a control terminal.

[0024] Each nanopore structure in the nanopore structure array can be considered as a pixel, each pixel including an orifice, an electric transducer element and a control terminal. The pixel array can be arranged as a linear grid in a manner similar to the arrangement of pixels on a television screen. When present in the nanopore structure, the nanopore forms a portion of the passage, i.e., a section of the passage with a nanometer width. The nanopore can be a solid-state nanopore, i.e., an orifice with a nanopore width is provided in a solid support. Alternatively, the nanopore can be a hybrid nanopore, wherein a biological nanopore is provided in the orifice of a solid support. The biological nanopore can be supported in an amphiphilic membrane. The amphiphilic membrane can be supported by a column as disclosed in WO2014 / 064443. The nanopore structure capable of supporting the nanopore can include an orifice with a width greater than the nanopore size (e.g., micrometer size). The nanopore structure can include a device for supporting the amphiphilic membrane therethrough. The analyte reservoir can be used to store analytes (e.g., analytes) for analysis. The analytes can pass through the nanopores in the nanopore sensor of the array. After passing through the nanopore, the analyte can remain in the pathway or flow out of the pathway into the outlet chamber. When the analyte reservoir, the outlet chamber, and the pathway of the nanopore structure array are provided with fluid, the drive electrode can impose a potential difference across the pathway. The drive electrode can provide a potential difference across the orifice to induce the charged analyte to pass through the nanopores of the array. The potential difference can be varied to change the speed or direction of displacement of the analyte.

[0025] Each electrical transducer element in the array acts as a sensor electrode. Changes in the ionic current passing through the nanopore cause fluctuations in the potential caused by the changes in ionic current, which can be measured to determine the presence or nature of the analyte. The fluid in the device (which may be aqueous) may contain ions. Multiple analytes may be displaced.

[0026] The drive electrode is used to provide a common potential difference across the nanopore array, wherein multiple analytes can be measured simultaneously in the array. Measurements are made at the electrical transduction element in each nanopore structure.

[0027] In some embodiments, each nanopore structure can have an associated control terminal. Such a control terminal can be an independent connection to a control signal generated external to the structure. This allows the application of a potential independently of changing the potential difference across other nanopore structures in the array.

[0028] The control signal may be generated within the nanopore structure in response to an external trigger or switch. Alternatively, the control signal may be generated from a circuit internal to the nanopore structure. The control signal has the effect of changing the voltage level at each nanopore structure. The control signal may be applied by the electrical transduction element to modify the voltage between the pathway and the drive electrode. Additionally or alternatively, the control signal may be applied via an electrical connection in the pathway, such as a control terminal or a further control electrode.

[0029] The device may have a single drive electrode disposed in electrical connection with the analyte reservoir and a single drive electrode disposed in electrical connection with the outlet chamber, wherein the drive electrodes are used to provide a common potential difference across the nanopore array.

[0030] Alternatively, the device may include multiple drive electrodes on the cis and / or trans side of the device.

[0031] Applying a control signal to an individual nanopore structure can act to change the potential difference across the nanopore structure and between the nanopore structure and the drive electrode. For example, the drive electrode in the analyte reservoir can have a voltage level of -0.1 volts, while the drive electrode in the outlet chamber can have a voltage level of 0.2 volts, resulting in a potential difference of 0.3 volts across the pathway of the array. Applying a control signal to impose a voltage of -2 volts at the nanopore structure results in potential differences of -1.9 volts and -1.8 volts between the nanopore structure and the cis electrode and between the nanopore structure and the trans electrode, respectively.

[0032] The electrical transduction element and the control terminal of each nanopore can be directly connected. In doing so, the electrical transduction element can serve as both a sensor electrode and a control electrode. This can be implemented by providing an electrical transduction element with two terminals: one for connecting to the sensing circuitry and the other for connecting to the control circuitry. In practice, the sensing circuitry and the control circuitry can reside in the same circuit or component. Any circuit can be located outside the structure and connected to the structure by, for example, wire bonding.

[0033] The control terminals may be configured to apply a control signal in response to a measurement by the electrical transduction element of a fluid potential at the electrical transduction element of each corresponding nanopore structure to change a potential difference from the drive electrode to the nanopore structure. The application of the control signal may be configured to change a potential difference between at least one of the control terminals and at least one of the drive electrodes.

[0034] A control signal applied to the control terminal of the nanopore structure can change the magnitude and / or polarity of the potential difference between the nanopore and the drive electrode, which can change the rate of passage of the analyte through the nanopore structure or change the direction of movement of the analyte.

[0035] The control signal may be connectable to a plurality of the nanopore structures to simultaneously change the potential difference between the connected control terminal and at least one of the drive electrodes.

[0036] The control signal can be applied for purposes other than rejecting the analyte or controlling the speed and / or direction of its displacement. For example, the control signal can be applied to induce the insertion of a biological nanopore in a membrane supported by the nanopore structure. The electrical transducer element can be connected to a measurement circuit to read the signal received from the electrical transducer 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 system and prevent the control signal from affecting the performance of the measurement circuit system.

[0037] In other words, the electrical transduction elements may be isolated prior to said applying of the control signal.Each individual electrical transduction element of each nanopore structure may be selectively isolated prior to applying the control signal.

[0038] The control signal may be applied for various purposes.

[0039] The control signal may be applied independently of the measurement result of the analyte.For example, the control signal may be applied to a membrane supported by the nanopore structure to induce insertion of a biological nanopore in the membrane.

[0040] The control signal may be applied to the nanopore structure in response to measurements made by the electrical transduction element.

[0041] For example, when the device determines that a passage through a nanopore is blocked by, for example, an analyte, the control signal may be applied for the purpose of unblocking the nanopore.The control signal may then be applied to unblock the passage.

[0042] The device can determine that the nanopore is blocked by measuring the change in the potential caused by the inhibition of current passing through the nanopore. In the absence of an analyte interacting with the nanopore, the ionic current passing through the nanopore due to the presence of ionic salts in the aqueous sample can be referred to as an open pore current. When the analyte interacts with the nanopore, the ionic current passing through the hole decreases and the change in ionic current reduction can be measured as the fluctuation of the potential at the sensor electrode when the analyte (such as DNA) shifts the nanopore. For example, the blockage of the nanopore caused by the analyte becoming fixed in the hole can cause the ionic current to decrease, and its value changes very little over time. In another example, the control signal can be applied for ejecting an analyte that is not interested or no longer interested from the nanopore. Measurement can be performed in real time so that a decision to eject the analyte can be made before a comprehensive measurement of the analyte, such as a decision to eject polynucleotides.

[0043] Regarding the previously mentioned devices for sequencing polynucleotides, such as the MinION TM The device is a device in which the current passing through the nanopore is measured by applying a potential difference between a corresponding electrode array on one side of each nanopore provided in the nanopore and a common electrode on the other side of the nanopore provided in the analyte reservoir. Because each nanopore has an associated electrode, it is possible to individually control the potential difference across each nanopore in the array and eject the analyte. In the embodiments described below, various advantages associated with measuring the local potential at each nanopore by means of an electrical transducer element are demonstrated. The drive electrode is used to provide a potential difference across the nanopore array, rather than to measure the analyte. Therefore, it is not possible to individually control the potential difference at the nanopore by the drive electrode. However, it is possible to individually control the potential difference across each nanopore by means of a control terminal.

[0044] The array of nanopore structures may include circuits, each circuit being associated with a respective nanopore structure and connected to the electrical transduction element. Each circuit may be configured to modify and / or process a signal received from the electrical transduction element. The circuit may also apply a control signal to the electrical transduction element. The circuit may isolate the control signal applied to the electrical transduction element from other sensing and processing functions.

[0045] Each circuit can reside within a pixel of the nanopore structure. Each circuit can be addressable. Each nanopore structure can be addressable. Addressing functionality can allow an external processor to communicate with the nanopore structure to perform at least one of: receive measurement information or control the movement of an analyte in a pathway. In this way, the measurement and control of sensing at each individual pathway can be individually controlled. The circuit can be disposed on or embedded within a support structure.

[0046] 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 controlling the nanopore structures in the group can be multiplexed. In this way, the electronic circuit can be addressable and can use multiplexing to control individual nanopore structures.

[0047] Each circuit may be associated with a respective nanopore structure or a group of nanopore structures. Each circuit may be connected to the control terminal and / or the electrical transduction element such that the circuit is configured to change the potential imposed by the drive electrode at the respective nanopore structure in response to measurements at the electrical transduction element and / or from an external processor attached thereto.

[0048] The structure may have a nanoporous layer incorporating nanopores and / or incorporating pores for supporting a solid film or a membrane having nanopores. When provided with nanopores, the nanopore structure may operate as a nanopore sensor. After the nanopore structure has been formed, the nanopore layer may be provided with nanopores. Nanopores may be provided by a user after a device having a nanopore structure has been provided to it. The nanopore layer may be replaced so that the device is recyclable. The nanopore structure may also include a base layer incorporating a channel. The nanopore layer and the base layer may be sandwiched or laminated together so that the nanopores and / or the pores are aligned to define the passage. At least one of the electric transducer element, the circuit or the control terminal is disposed on or between the outer surface of the structure. A separate nanopore structure may include a single structure or one or more substructures connected to each other. A single structure or substructure may be planar or sheet-like.

[0049] Each nanopore structure can be defined by its passageway. The passageway can fluidically connect cis and trans states. The passageway can be formed by a formation within each nanopore structure, for example, formed by: a nanoporous layer for supporting the nanopores, the layer having through-holes; and a base layer having channels, the base layer serving as the through-holes. The through-holes of the nanoporous layer and the base layer are aligned to form a passageway.

[0050] The electrical transduction element defines a portion of the pathway. For example, the electrical transduction element may be sandwiched or laminated between the nanoporous layer and the base layer. However, the electrical transduction element may be positioned elsewhere in the pathway. If there is a fluid connection between the electrical transduction element and the nanopores disposed in the nanoporous layer, and a direct fluid connection is possible, the electrical transduction element may be configured to surround the pathway.

[0051] The electrical transduction element and / or the circuit can be implemented on a sensing layer. The sensing layer can be a substructure. The sensing layer can be sandwiched or embedded between the nanoporous layer and the base layer, with the through-holes of the sensing layer aligned with the through-holes of the nanoporous layer and the base layer. Specifically, the nanoporous layer, the sensing layer, and the base layer can be a substructure that is stacked to provide a nanoporous structure array.

[0052] When disposed in the nanopore structure, the nanopore forms part of a pathway. Rejection of an analyte can be managed using a control signal, which serves to control the movement of the analyte within the nanopore, such as rejecting the analyte from the nanopore. The nanopore in the pathway may become blocked. Blockage of the nanopore can be sensed, and a control signal can be applied to the nanopore structure to clear the blockage.

[0053] The nanopore can be a solid-state nanopore, that is, a hole of nanometer width arranged in a solid-state membrane. Such a membrane can be a nanoporous layer, or a membrane placed on a nanoporous layer. The solid-state nanopore can be located on the nanoporous layer. Alternatively, the nanopore can be a biological nanopore located in a solid-state film or membrane. Further alternatively, the nanoporous layer can be formed with holes, and a membrane (such as an amphiphilic membrane or a lipid bilayer) can be formed across the holes so that the nanopore can be inserted into the membrane. In each of these nanopore examples, each nanopore structure in the array can be provided with a nanopore.

[0054] The inventors have also sought to improve the architecture of nanopore sensors, particularly where such improvements could optimize sensitivity and performance. Generally, the inventors have attempted to achieve this by providing an architecture with nanopore structures, wherein the nanopore structures positioned within the architecture provide fluid communication from one side of the structure to the other via pathways provided within each nanopore structure. In this way, the architecture can separate cis from trans. Each nanopore structure within the nanopore architecture has a sensor electrode. To minimize attenuation of the signal derived from the sensor electrode and avoid any noise impairment of the signal, each nanopore structure is provided with circuitry for processing the signal from the sensor electrode before the processed signal is transmitted for further processing and / or analysis. The circuitry can be embedded within the nanopore structure. The circuitry can occupy the same footprint as the nanopore structure, allowing the nanopore structure to be considered an active pixel. Nanopore structures with their own circuitry can complement the improved control mechanisms disclosed herein by locally generating and applying control signals, thereby minimizing the impact of the control signals on other nanopore structures in the array.

[0055] Thus, in a second aspect, the present invention provides a device for sensing an analyte having a nanopore structure, the nanopore structure being arranged to separate an analyte reservoir from an outlet chamber, each nanopore structure providing a passage for fluidly connecting the analyte reservoir and the outlet chamber through the structure, wherein each nanopore structure comprises: an electrical transduction element; and an electronic circuit configured to detect and optionally amplify a signal from the electrical transduction element, wherein each of the structures is configured to perform one or more of: store at least a portion of the signal; transmit the at least a portion; process the at least a portion; and communicate the at least a portion to a connectable processor.

[0056] The nanopore structure may be included as part of an overall structure, wherein individual nanopore structures are connected to each other.

[0057] The structure can be configured to separate an analyte chamber for receiving an analyte from an outlet chamber for collecting the analyte. Drive electrodes can be connected to the analyte reservoir and the outlet chamber, respectively, to impose a potential difference across the pathway in the nanopore structure. When provided with a nanopore, the nanopore structure can function as a nanopore sensor, and the device can be a nanopore sensing device.

[0058] Each of the nanopore structures in the array may further include a compensation circuit. The functionality of the compensation circuit may be combined with other processing functions of the circuitry in the nanopore structure. The compensation circuit may have a variable gain amplifier and / or a variable capacitor in a feedback loop of the compensation circuit.

[0059] As described in the first aspect, the structure may have a control terminal to apply a control signal to vary the potential difference across the nanopore structure. The control signal may be switchably applied to the control terminal to adjust a configurable voltage level imposed on the pore.

[0060] The nanopore structure in combination with the circuitry (which may include compensation circuitry) may be packaged within a defined footprint or pixel space.The array of pixel-spaced nanopore structures may be arranged in a tessellated array.

[0061] By processing the signal from the electrical transducer element at least partially within the nanopore structure itself, the signal can be processed or managed locally. For example, the signal can be amplified locally so that the attenuation or noise affecting the signal is minimized before the signal is analyzed elsewhere. The circuit can also store the signal, signal value, or data derived from the signal. In this way, information derived from the nanopore structure can be transmitted to a processor remote from the nanopore structure as needed. Each nanopore structure or circuit in the nanopore structure can be addressable. The circuit can be connected to an analog-to-digital converter (ADC) located outside the nanopore structure.

[0062] Furthermore, the inventors have attempted to provide a structure that generally improves the manufacturability of nanopore structure arrays while improving sensitivity and performance. The nanopore structure arrays herein can not only provide improved nanopore structures, but also can complement the integration of control functions and local control.

[0063] Therefore, in a third aspect, the present invention provides a device having an array of nanopore structures. The structure can be configured in a thin sheet, the thin sheet comprising: a nanopore layer having an array of nanopores and / or an array of holes 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 thin sheet, wherein the nanopores and / or holes are aligned with the channels, wherein each nanopore structure in the nanopore structure comprises a passage, each passage being defined at least in part by: one of the nanopores and / or one of the holes of the nanopore layer on one side of the passage; a channel of the base layer on the other side of the passage; and an electrical transduction element.

[0064] In another aspect, the present invention provides the nanopore structure array itself. When provided with nanopores, each nanopore structure of the array functions as a nanopore sensor. Each nanopore structure has a through-hole defined by a nanopore (if provided) or a hole, a channel, and an electrical transduction element.

[0065] The thin sheet can be a substantially planar array of nanopore structures. When the nanopore structure is provided with nanopores, the nanopore structure can be used as a nanopore sensor. The thin sheet can be configured in the device to separate the analyte reservoir from the outlet chamber. The analyte reservoir and the outlet chamber can contain fluid. The passage can be filled with fluid and provide a fluid connection between the analyte reservoir and the outlet chamber.

[0066] Configuring the nanopore layer and the base layer as separate layers can improve the scalability of the sheet. The layers can facilitate the assembly of the device, thereby reducing manufacturing costs. The layering of the sheet can bring the components of the nanopore structure together in an efficient manner. In addition, by having different components of the nanopore structure on different layers, the formation or configuration of the components can be optimized. Often, the process used in the manufacture of one component is incompatible with or detrimental to the manufacture of another component. In addition, the optimal material for forming one component can be different from the optimal material for forming other components. For example, the nanopore array and / or the pore array of the nanopores can be formed separately from the base layer. The nanopore layer and the base layer can include different materials. The separate layers can enable the components of the nanopore structure to be optimally configured and / or positioned.

[0067] The provision of the layer may enable the layer to be replaced. The nanoporous layer may be removably attached. In this way, the nanoporous layer may be replaced by a replacement nanoporous layer so that the device can be recycled if, for example, the nanoporous layer becomes contaminated.

[0068] Each nanoporous structure of the sheet is defined by the passage. The individual components of the nanoporous structure (i.e., the nanopore or nanoporous hole, the electrical transduction element, and the channel) form the passage. The nanoporous layer need not have nanopores and may be provided with nanopores. The nanopore may be arranged above the hole of the nanoporous layer, and in doing so, the additional nanopore above the hole also forms an element of the passage.

[0069] The electrical transduction element in each passage may be positioned between the nanopore layer and at least a portion of the channel. The electrical transduction element may be configured with a connection for measuring the electrical potential of the fluid at the location of the electrical transduction element when the structure is provided with nanopores and a fluid is provided in the passage.

[0070] By connecting the fluids in the cis and trans pathways, the electrical transduction element can produce a characteristic indicative of the potential of the fluid in the pathway at the electrical transduction element. The electrical transduction element can be an electrical connection. The electrical transduction element can be located in a cis or trans reservoir, on a surface of the nanopore structure, at a location within the pathway, or at another location within the nanopore structure.

[0071] The electrical transduction element may be a device or region of a device and / or a combination of circuits, conductors or circuit elements that senses the fluid potential at the electrical transduction element of the device. Additionally or alternatively, the circuit may be provided as a transduction element to generate a signal indicative of the local potential.

[0072] As described above, the device can have an analyte reservoir and an outlet chamber separated at least in part by the sheet. The analyte reservoir can be used as a cis when provided with a sensor and hold an analyte to be analyzed by the nanopore structure. The passageway of 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 passageway, or more specifically, a nanopore in a nanopore sensor (i.e., a nanopore structure provided with a nanopore).

[0073] The device may have a drive electrode connected in the analyte reservoir and the outlet chamber to impose a potential difference across the array of pathways between the analyte reservoir and the outlet chamber.

[0074] The sheet can be substantially planar. As the structure incorporating the nanopore structure array, the surface of the sheet can have a cis surface on the nanopore layer that faces the analyte reservoir and defines a cis plane, and a trans surface of the base layer that faces the outlet chamber and defines a trans plane. The array of electrical transduction elements can be embedded within the sheet at least partially between the cis plane and the trans plane. The electrical transduction elements of the array can be sandwiched between the nanopore layer and the base layer.

[0075] Each nanopore structure of the array may have a hole formed at a first end of the passage. A nanopore may be configured at the first end of each hole. The electrical transducer element may be configured on a side of the hole opposite the nanopore. The size of the hole may be larger than the size of the nanopore and increase the volume of the fluid surrounding the nanopore. Specifically, the diameter of the hole may be larger than the diameter of the nanopore. The nanopore may reside in a membrane spanning the hole. The membrane may be a solid membrane, an amphiphilic membrane, or a lipid bilayer. The nanopore defines a portion of the passage. Entry and exit from the hole are through the nanopore and a hole outlet.

[0076] The pores can be configured to support a fluid membrane, such as a polymer membrane or a lipid bilayer. The nanoporous layer can be made of a different material than the base layer. By using different materials for the nanoporous layer, a material can be selected that has a surface energy that optimizes the formation of a membrane across the pores that support the nanopores.

[0077] The electrical transduction element may be a sensor electrode. The sensor electrode can be directly connected to the base or gate of a transistor device to measure a change in the potential of the fluid at the location of the electrical transduction element when a fluid is provided in the passage. As described herein, a nanopore structure provided with a nanopore forming part of the passage serves as a nanopore sensor, and sensing is performed by the electrical transduction element.

[0078] The electrical transduction elements of the nanopore structures of the array may be connected to edge connectors or wire bonds. The connectors may provide connections to measurement circuitry external to the wafer (i.e., separate from the array of nanopore structures). The connectors may be connected to vias that lead to connections at the edge of the wafer for subsequent connection to measurement circuitry external to the wafer. The transistor devices may be field effect transistors.

[0079] The sheet has so far been described as having a nanoporous layer and a base layer. The electrical transduction element may be a layer within the sheet, or may have elements sandwiched between layers. However, the sheet of the device may further include a sensing layer having an array of the electrical transduction elements, wherein the sensing layer is sandwiched between the nanoporous layer and the base layer. The electrical transduction elements may be formed on the sensing layer. The electrical transduction elements may have an exposed portion for connecting to the fluid in the passage and an embedded portion embedded in the sheet. Additionally or alternatively, the electrical transduction elements may have a connection portion for connecting to a measurement circuit separate from the sheet. By incorporating the electrical transduction elements in or on the sensing layer, this enables the formation of the electrical transduction elements to be separated from the manufacture of the other layers. The sensing layer may be manufactured using different materials, processes and / or techniques than the other layers.

[0080] The electrical transducer element may at least partially cover the wall of the passage. The electrical transducer element may cover a portion of the wall of the channel in cross section. The electrical transducer element may form a ring around the base of the passage and / or a hole or cavity within the passage.

[0081] The electric transduction element may be formed on one surface of the sensing layer. The sensing layer may be sandwiched between the base layer and the nanoporous layer, wherein the electric transduction element is aligned with the nanopores or the holes of the nanoporous layer and the channels of the base layer. When aligned, the face of the sensing layer may expose the electric transduction element to the nanoporous layer, such that the nanoporous layer is formed or placed on the surface having the electric transduction element; in this arrangement, the electric transduction element may be said to face the nanoporous layer. Alternatively, when aligned, the face of the sensing layer may expose the electric transduction element to the base layer, such that the electric transduction element is formed or placed on the surface of the base layer; in this arrangement, the electric transduction element may be said to face the base layer.

[0082] The electrical transducer element at least partially surrounds the passageway, forming the surface of the sensing layer and having an exposed portion disposed facing the outlet chamber. The exposed portion may form a portion of a wall of a cavity formed in the sensing layer between the hole and the channel. The cavity allows a larger area of ​​the sensor electrode to be exposed to the fluid in the passageway. This can improve the sensitivity of the sensor electrode.

[0083] The electric transduction element may have an aperture forming part of the passage and an exposed portion, wherein in cross section, a ratio of a size of the exposed portion of the electric transduction element to a size of the aperture is 1: 1. The ratio may be approximately 5:1.

[0084] The electric transduction element may have an aperture forming a portion of the passage and an exposed portion, wherein in a plan view, a ratio of a size of the exposed portion of the electric transduction element to a size of the aperture is 1:1. The electric transduction element may have an aperture forming a portion of the passage and an exposed portion, wherein the ratio is approximately 5:1. The aperture may be circular.

[0085] The electrical transduction element may have a larger exposed area to increase exposure to the fluid in the passageway, thereby increasing the element's sensitivity to voltage fluctuations caused by analytes passing through or through the nanopore in the passageway.

[0086] The sensing layer may incorporate electronic circuitry for each nanopore structure. The circuitry may be connected to the electrical transduction element to modify and / or process the signal received from the electrical transduction element. By incorporating electronic circuitry within each nanopore structure, the signal from the electrical transduction element can be processed locally to prevent any attenuation of information in the signal derived therefrom and / or prevent any degradation of the signal by noise. Each circuit in the corresponding nanopore structure may process the signal from the sensor electrode before the processed signal is transmitted outside the sheet for further processing and / or analysis. By incorporating the circuitry in the sensing layer, the circuitry may be embedded within the nanopore structure. The circuitry may occupy the same footprint as the nanopore structure, allowing the nanopore structure to be considered an active pixel. Nanopore structures with their own circuitry can complement the improved control mechanisms disclosed herein by locally generating and applying control signals, thereby minimizing the impact of the control signals on other nanopore structures in the array. The circuitry within the sensing layer of the nanopore circuitry may be a compensation circuit.

[0087] The electronic circuit may be configured to detect a change in resistance at a nanopore when an analyte passes through or is adjacent to a nanopore in a corresponding pathway.The circuit may detect a change in resistance detected by a fluid in the sensor.

[0088] Although the device has been described as being suitable for sensing an analyte, it will be appreciated that the analyte is one that can be measured using a nanopore. For example, the analyte may be a protein, a polymer, a polynucleotide, or the like.

[0089] The electronic circuit can detect a change in resistance at the nanopore when the polymer passes through the nanopore, convert the change in resistance into a voltage signal, and amplify the voltage signal. The electronic circuit can filter the signal. The electronic circuit can sample and / or digitize the signal obtained from the electrical transducer element.

[0090] Each nanopore structure can have a plurality of electrical transducer elements corresponding to each corresponding nanopore structure. Similarly, each nanopore structure can have a plurality of circuits corresponding to each corresponding nanopore structure and / or the electrical transducer elements disposed in the nanopore structure. Each of the electrical transducer elements and / or circuits can be configured as 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.

[0091] The array of nanopore structures may be connected to an architecture for enabling readout from each nanopore structure (which may be referred to as a pixel) individually in the matrix array.Each nanopore structure may have a row and column number.

[0092] Each electrical transduction element may have dedicated electronic circuitry, and each electrical transduction element and electronic circuitry may be positioned in a footprint. The footprint may be a pixel, such that the nanopore structure is tessellated in the array.

[0093] While each nanopore structure of the array has an electrical transduction element and optionally circuitry and / or control terminals, it will be appreciated from the teachings herein that each nanopore structure can have multiple electrical transduction elements and / or multiple circuits, each circuit providing one or more functions. For 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 the passageway in the nanopore structure, the second electrical transduction element having circuitry for controllably applying the control signal.

[0094] Thus, multiple electrical transducer elements can be arranged in a module having multiple corresponding nanopore structures. The module can have a common dedicated electronic circuit, and each of the electrical transducer elements and the electronic circuit are positioned in the footprint occupied by the multiple nanopore structures. The module can have, for example, four nanopore structures, each with a corresponding electrical transducer element, wherein each electrical transducer element is connected to a common circuit. The common circuit can be addressably connected to an external off-structure or off-sheet electronic circuit.

[0095] The plurality of nanopore structures may be arranged in a two-dimensional matrix. The plurality of nanopore structures may be arranged in a checkerboard pattern.

[0096] The electric transducing element may be connected to a base or a gate of a transistor for sensing. The transistor may be a field effect transistor.

[0097] Each of the nanopore structures may have a control terminal for applying a control signal to change the potential difference across the corresponding nanopore structure. The control terminal may be switchably connected to the electrical transducer element. The control terminal may be switchably connected to a power source to change a configurable voltage level imposed on the pore. The electrical transducer element and the connection for measuring the potential of the fluid may be switchably isolated from the control signal. The electrical transducer element and the control electrode may be physically separate. At least a portion of the electrical transducer element and at least a portion of the control electrode may extend in the same plane. At least a portion of the electrical transducer element and at least a portion of the control electrode may at least partially form the base of the pore. At least a portion of the electrical transducer element and at least a portion of the control electrode may extend perpendicular to each other. At least a portion of the control electrode may be at least partially disposed in the channel. The surface area of ​​the electrical transducer element exposed to the channel may be smaller than the surface area of ​​the control electrode exposed to the channel.

[0098] The device described herein can be configured with a conductive guard disposed in at least one of the nanoporous layer, the base layer, or the sensing layer. The conductive guard can extend between at least one of the electrical transduction element and a signal conductor connected to the electrical ring energy element and a parasitic conductive element in the nanoporous layer, the base layer, or the sensing layer to prevent parasitic capacitance from affecting measurements obtained from the connection. A buffered version of the input signal can be applied to the guard conductor. Thus, no voltage difference exists across the capacitance from the input signal conductor to the conductive substrate.

[0099] The conductive guard may at least partially comprise an insulated guard conductor having an insulating layer.The conductive guard may be configured to extend at least partially between the base layer and the channel.

[0100] The inventors have further considered the operation and manufacturability of the devices disclosed herein.

[0101] In a further aspect, the present invention provides a method of operating an apparatus for performing nanopore sensing as described, the method comprising: causing an analyte to transduce through an array of nanopores under application of a potential difference across the array; measuring a change in fluid potential at each nanopore by means of a corresponding electrical transduction element and responding to the measurement; applying a control signal to a control terminal of the electrical transduction element to change the potential difference across the nanopores. Thus, in a further aspect, the present invention provides a method of operating an apparatus for performing nanopore sensing, the method comprising: imposing a potential difference across an array of nanopore sensors disposed in a structure separating an analyte reservoir from an outlet chamber, each nanopore sensor having a passageway for providing a 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 transduction element for measuring a change in fluid potential at the electrical transduction element of the nanopore sensor when the analyte is induced to pass through a nanopore of the nanopore sensor; and applying a control signal to the control terminals of the electrical transduction elements of the nanopore sensors of the array to change the potential difference across the nanopore sensors. The fluid potential can be measured at the electrical transduction element. When the device is provided with a fluid, the electrical distribution of the fluid across the nanopore structure can be changed. In operation, the fluid resides 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.

[0102] The potential difference imposed across the array is used to induce the analyte to pass through the pathway or at least into the pathway. The analyte to be analyzed is provided in the analyte reservoir and induced to the outlet chamber by the drive electrodes. However, this situation can be reversed, in that the analyte can be provided in the outlet chamber, or the analyte in the outlet can be induced into the analyte reservoir by the drive electrodes, for example by changing the potential difference between the drive electrodes.

[0103] In each case, the electrical transduction element of each nanopore structure (which is provided with a nanopore to serve as a nanopore sensor) can measure changes in the potential of the fluid. The dimensions of the nanopore structure array are set so that the electrical transduction element of one nanopore sensor is prevented from detecting an analyte that has passed through a nanopore in an adjacent nanopore structure.

[0104] A control signal may be applied to the element to change the potential difference across the nanopore sensor in which the element resides.

[0105] The control terminal connected to the electrical transduction element can be switchably connected to the control terminal of the electrical transduction element to apply the control signal to the electrical transduction element. Additionally or alternatively, the device can be operated to isolate any sensing circuitry from the electrical transduction element to prevent damage to the circuitry when the control signal is applied.

[0106] The method may include analyzing a characteristic of a localized change in electrical potential at the nanopore sensor and applying the control signal to the nanopore sensor in response to a predetermined characteristic. The method applies the control signal to an electrical transduction element of the nanopore sensor to change the electrical potential difference imposed by the drive electrode at the nanopore sensor. The change in electrical potential difference may induce movement of an analyte or a freely movable nanopore that may carry an electrical charge.

[0107] The control signal can perform a variety of operations, including but not limited to: inducing pore insertion into a membrane formed across the pathway; unblocking the nanopore; rejecting analyte; and altering the rate at which analyte translocates through the nanopore.

[0108] In forming a device having a nanopore structure for sensing an analyte, the formation method includes: forming a nanopore structure in a structure and arranging the structure to separate an analyte reservoir from an outlet chamber of the device, so that each nanopore structure provides a passage for fluid connection between the analyte reservoir and the outlet chamber through the structure; and manufacturing the following in each nanopore structure: an electrical transduction element; and an electronic circuit configured to measure a signal from the electrical transduction element, wherein each nanopore structure in the nanopore structures is configured to perform at least one of the following: store at least a portion of the measured signal or information derived from the at least a portion; transmit the at least a portion or the information; process the at least a portion or the information; and transmit the at least a portion or the information to a connectable processor.

[0109] Fabricating an electronic circuit in each nanopore structure may enable measurements to be performed at the electrical transduction element at the nanopore structure when the nanopore structure is provided with a nanopore for use as a sensor.

[0110] While measurements acquired from sensors can be directly transmitted to circuitry outside the structure for analysis, the ability to locally process or condition the signals or information therefrom can improve noise performance, data management, or amplification. For example, circuitry located within the nanopore structure can amplify signals received from the electrical transducer element, and by locally amplifying the signal, the amplified noise level is minimized. For example, if the signal received from the electrical transducer element were transmitted outside the structure for analysis before amplification, the signal's exposure to noise would increase and subsequently be amplified, thereby reducing the signal-to-noise ratio.

[0111] The method may further comprise configuring an analyte reservoir for receiving an analyte and an outlet chamber for collecting the analyte, and configuring the nanoporous layer to separate the analyte reservoir from the outlet chamber. The structure may separate the cis and trans forms of the device.

[0112] The method may further include configuring a drive electrode, the drive electrode being connected to the analyte reservoir and the outlet chamber, respectively, to impose a potential difference across the passageway of the nanopore structure. The imposed potential difference may be common across the plurality of nanopore structures. A plurality of drive electrodes may be provided to achieve a common potential difference across the array of nanopore structures.

[0113] The method may further comprise configuring the electronic circuitry with switchable connections for applying signals to respective control terminals of the electrical transduction elements to vary the electrical potential imposed by the drive electrode across each respective nanopore structure.

[0114] The method may further include forming a control electrode in the pathway of each nanopore sensor, the control electrode selectably connectable to a signal for varying the potential imposed by the drive electrode across each respective nanopore structure.

[0115] In terms of manufacturing a device having a nanopore structure for sensing an analyte, the manufacturing method includes forming a device having an array of nanopore structures configured in a thin sheet, the method comprising arranging the thin sheet to separate an analyte reservoir from an outlet chamber of the device, so that each nanopore structure provides a passage for fluid connection between the analyte reservoir and the outlet chamber through the structure, the method comprising: forming a nanopore layer having an array of nanopores and / or an array of support structures (such as holes) 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 or laminated to the nanopore layer to form the thin sheet, so that the nanopores and / or the holes are aligned with the electrical transduction elements and the channels; and providing a passage through each nanopore structure in the nanopore structure, such that each passage is at least partially defined by: one of the nanopores and / or one of the holes of the nanopore layer on one side of the passage; a channel of the base layer on the other side of the passage; and an electrical transduction element.

[0116] Aligning the nanoporous layer, the base layer, and the array of electrically transducing elements may include sandwiching the array of electrically transducing elements between the nanoporous layer and the base layer. The sandwiching step may include bonding or otherwise connecting the two layers.

[0117] The method may further comprise forming a cavity adjacent to at least a portion of each of the electrical transduction elements. The cavities may increase the area of ​​the element exposed to the fluid in the passageway.

[0118] The method may further include: forming the electric transduction element array on a sensing layer; and sandwiching the sensing layer between the nanopore layer and the base layer.

[0119] The method may further include: forming the array of electric transduction elements on a sensing layer; fabricating an array of electronic circuits in the sensing layer, the circuits being connected to corresponding electric transduction elements to modify and / or process signals received from the electric transduction elements; and sandwiching the sensing layer between the nanoporous layer and the base layer.

[0120] The method may further include arranging the electrical transduction element to have: (i) an exposed portion for connecting to fluid in the passage; and (ii) an embedded portion embedded within the structure; and / or (iii) a connection portion for connecting to a measurement circuit separate from the structure.

[0121] The method may further include forming a conductive guard in at least one of the nanoporous layer, the base layer, or the sensing layer, the 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 in at least one of the nanoporous layer, the base layer, or the sensing layer to prevent parasitic capacitance from affecting measurements obtained from the connection.

[0122] The method may further comprise providing each nanopore structure with a buffer connecting an output of the electrical transduction element of the nanopore structure to an electrically conductive protection device.

[0123] The method may further comprise providing an amphiphilic membrane in each of the nanopore structures of the array and inserting a biological nanopore in the membrane.

[0124] The method may comprise removably attaching the structure and / or removing the nanoporous layer and replacing the nanoporous layer with another nanoporous layer. In this way, the device may be recycled.

[0125] Many aspects have been described herein, and elements of different aspects may be combined according to the teachings herein. Thus, many additional aspects are implicit in accordance with the teachings of the description and the accompanying drawings, which typically combine two or more of the aspects described herein. In general, the different aspects may be combined in any combination.

[0126] Embodiments of the invention will be discussed below, by way of non-limiting examples only, with reference to the accompanying drawings, in which:

[0127] Figure 1 is a cross-sectional view of a known nanopore sensor;

[0128] Figure 2 is a cross-section of a single sensor electrode and corresponding biological nanopore within a nanopore array configured in a structure and connected by wires to a measurement circuit;

[0129] FIG3( a ) is an alternative cross-section of a single sensor electrode and corresponding biological nanopore, the sensor electrode being disposed on a sensor layer sandwiched between a nanopore layer and a base layer within a portion of a structure of an array of nanopore structures, wherein the sensor electrode is connected to an electronic circuit via a wire, while FIG3( b ) is equivalent to FIG3( a ) in which the sensing layer incorporates the electronic circuitry;

[0130] FIG4( a ) is a schematic diagram of the layout of the nanopore sensor of FIG3( b ), indicating the position of the pore relative to the electronic circuitry;

[0131] Figure 4(b) shows two adjacent sensor electrodes before the addition of the nanopore;

[0132] Figure 4(c) and 4(d) is an example of a portion of the structure having the nanopore sensor array of FIG4( a );

[0133] Figure 4(e) is a schematic diagram showing how structure may be arranged to separate two chambers in a device;

[0134] Figure 4(f) shows an alternative layout of four nanopore structures;

[0135] Figures 5(a) to 5(d) The various electronic circuits that implement the electronic functions that interface with the electrical transduction elements are shown;

[0136] Figures 6(a) to 6(e) is a schematic cross-sectional view of sensor electrodes and control electrodes configured in an array of nanopore sensors;

[0137] Figure 7(a) shows two schematic circuits illustrating the parasitic capacitance in an array with and without protection, while Figure 7(b) shows the Figure 2 An alternative cross-sectional view to the cross-sectional view shown in , in which the protective conductor is arranged in the structure and connected to the measuring circuit via a further conductor, and Figure 7(c) and 7(d) shows an alternative cross section in which protection is provided; and

[0138] Figure 8 and 9 is a side view of a corresponding example of a device comprising an array of nanopore structures.

[0139] The embodiments include corresponding components that are generally labeled with the same reference numerals. For clarity, descriptions of corresponding components are not repeated unless the context requires otherwise, but are generally applicable to all embodiments. Not every component is labeled in every figure, nor is every component of every embodiment of the present invention shown, where description is not necessary for understanding the present invention.

[0140] In summary, an apparatus for performing improved nanopore sensing is described. An example apparatus may have a structure arranged to separate an analyte reservoir from an outlet chamber. The structure may have an array of nanopore structures, each nanopore structure including a passageway for achieving a fluid connection between the analyte reservoir and the outlet chamber through the structure. A control terminal may be included, wherein each terminal is connected to a respective nanopore structure to apply a control signal to change the potential difference across the nanopore structure. In another aspect, an improved nanopore structure for sensing an analyte may include an electronic circuit configured to detect a signal from an electrical transduction element, and wherein each of the structures may be configured to perform at least one of: store at least a portion of the signal; transmit the at least a portion; process the at least a portion; and transmit the at least a portion to a processor.

[0141] Some embodiments of a device for improved nanopore sensing having an array of nanopore structures have an array of nanopore structures configured in a thin sheet, the thin sheet comprising: a nanopore layer having an array of nanopores and / or an array of holes for supporting nanopores; and a base layer having an array of channels, the base layer being sandwiched or laminated to the nanopore layer to form the thin sheet, wherein the nanopores and / or the holes are aligned with the channels, wherein each of the nanopore structures comprises a passage, each passage being defined at least in part by: one of the nanopores and / or one of the holes of the nanopore layer on one side of the passage; a channel of the base layer on the other side of the passage; and an electrical transduction element.

[0142] Aspects of the present invention further relate to a method of operating an apparatus for performing nanopore sensing, the method comprising applying a control signal to a control terminal of an electrical transduction element of a nanopore structure of an array to vary a potential difference across the nanopore structure.

[0143] Additional embodiments relate to methods of forming a device having a nanopore structure for sensing an analyte.Example methods may include fabricating in each nanopore structure: an electrical transduction element; and electronic circuitry configured to measure a signal from the electrical transduction element.

[0144] Figures 2 to 4(a) FIG1 is a cross-sectional view of a portion of a structure 100 having a nanopore structure incorporated therein. The structure 100 has an array of nanopore structures, each of which is adapted to support a nanopore 116. When configured with nanopores, the nanopore structure of the device can function as a nanopore sensor. The nanopore sensor 102 herein is a nanopore structure having a nanopore.

[0145] Figures 4(b) to 4(f) Shown Figures 2 to 4(a) The plurality of nanopore sensors 102 shown in FIG. 1 may be arranged as part of a nanopore structure array 104. Such an arrangement may be referred to as a two-dimensional matrix of nanopore structures or a nanopore sensor array.

[0146] The structure 100, which may take the form of a sheet, incorporates an array of nanopore structures 104 (note: only one nanopore sensor 102 of the array is shown) and may be configured within a device or apparatus for analyzing an analyte as shown in FIG. 4(e).

[0147] The structure 100 separates an analyte reservoir 106 for receiving an analyte from an outlet chamber 108. The structure 100 has a nanopore layer 110 disposed on a base layer 112, which together form at least a portion of the structure 100 having a plurality of nanopore sensors 102. Each nanopore sensor 102 in the array 104 has a passage 114 or fluid passage configured to extend through the nanopore layer 110 and the 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 referred to as an analyte chamber, a sample chamber, a cis, a cis reservoir, or a first fluid reservoir. The outlet chamber may also be referred to as a trans, a trans reservoir, or a second fluid reservoir.

[0148] The nanopore layer 110 of each nanopore sensor 102 can optionally be provided with a nanopore 116 in a membrane 118 supported by the nanopore layer 110. Alternatively, the nanopore 116 can be a so-called solid-state nanopore, i.e., a nanometer-sized through-hole disposed in a solid-state supporting layer. Further alternatively, the nanopore 116 can be a so-called hybrid nanopore, i.e., a biological nanopore disposed in an orifice in a solid-state membrane. In either case, the nanopore 116 is disposed in the membrane 118 near the first end 120 or pore end of the passage 114 (e.g., at the top of the sensor as shown).

[0149] The base layer 112 has a channel 122 near a second end 124 or channel end, which is the end of the channel 114 opposite the first end 120 (e.g., at the bottom of the nanopore sensor 102 as shown). The channel 114 extends through the nanopore structure 100, connecting one side to the other. The channel 122 forms a portion of the channel 114. The channel 122 is structurally and geometrically configured to act as a fluid resistor. This can be achieved by defining the aspect ratio of the channel 122. Additionally or alternatively, other techniques for implementing fluid resistance in the channel 122 can be used.

[0150] The fluid resistance of channel 122 can be changed by changing its dimensions (specifically, its aspect ratio) and by changing the ion concentration of the fluid in analyte reservoir 106 and outlet chamber 108. For example, channel 122 can be configured with a high aspect ratio to increase resistance. Additionally or alternatively, the fluid in channel 122 can have a lower ion concentration than the fluid in analyte reservoir 106 and outlet chamber 108 to increase the resistance of the channel. Maintaining a higher ion concentration in analyte reservoir 106 and outlet chamber 108 improves the signal-to-noise ratio.

[0151] In some embodiments, the aspect ratio can be, for example, between about 100:1 and about 2000:1, which is the ratio of the channel length to the channel diameter or largest lateral dimension.

[0152] In some embodiments, the ion concentration difference may be between about 1:1 and about 2000:1, such as around 1000:1, 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 .

[0153] The channel 122 can be configured such that the resistance of the channel 122 and the nanopore 116 are substantially matched when the passage 114 is occupied by fluid and are relatively high relative to the resistance of the fluid in the analyte reservoir 106 and the outlet chamber 108, so that the resistance of the analyte reservoir 106 and the outlet chamber 108 does not significantly affect the measurement. In other words, the channel 122 is configured as a fluidic resistor to approximate the resistance of the nanopore 116. This means that the resistance of other circuit elements (such as the fluid in the analyte reservoir 106 and the outlet chamber 108) is less important and does not need to be compensated for when making measurements.

[0154] The signal-to-noise ratio can be optimized by selecting the fluid resistance of channel 122 to be equal to the resistance of nanopore 116. However, this is not required, and the fluid resistance of channel 122 can be varied based on this value to take other factors into account while still achieving an acceptable signal-to-noise ratio. An acceptable signal-to-noise ratio can be achieved by having the fluid resistance of channel 122 significantly less than the resistance of nanopore 116, for example, having the fluid resistance of channel 122 be 10% or less of the resistance of nanopore 116, for example, 2% or less of the resistance of nanopore 116. In some embodiments, the lower limit of the fluid resistance of channel 122 can be set by the desired signal-to-noise ratio. In other embodiments, the lower limit of the fluid resistance of channel 122 can be set by the threshold of crosstalk between adjacent channels during a flick (as described below). That is, the fluid resistance of channel 122 is desirably significantly greater than the resistance from the end of channel 122 to the electrical transduction element to prevent these resistances from forming a voltage divider that imposes a fraction of the applied voltage on adjacent channels 122.

[0155] Other factors that may be considered in selecting the fluid resistance of channel 122 are as follows.

[0156] As the fluid resistance of channel 122 increases, ion diffusion decreases, resulting in increased ion depletion near the pore and, consequently, signal decay within the timescale of a typical event for signal acquisition. To increase the read length limit caused by this effect, the fluid resistance of channel 122 can be reduced. In many embodiments, this factor can set an upper limit on the fluid resistance of channel 122.

[0157] Because channel 122 and nanopore 116 act as a voltage divider, the voltage across nanopore 116 is affected by the current flowing through it. As the fluid resistance of channel 122 increases, the variation in the voltage across nanopore 116 increases, which can complicate signal processing. To limit this effect, the fluid resistance of channel 122 can be reduced.

[0158] Channels with lower fluidic resistance are easier to fabricate and could open up alternative manufacturing techniques that increase yields or reduce costs.

[0159] Reducing the fluid resistance of channel 122 may increase bandwidth or provide room for additional capacitance in passageway 114 .

[0160] Taking these factors into account, the fluid resistance of channel 122 can be less than the resistance of nanopore 116, typically at most 50% or at most 25% of the resistance of nanopore 116. In some embodiments, the optimal fluid resistance of channel 122 can be about 10% of the resistance of nanopore 116.

[0161] When the ratio of the fluid resistance of channel 122 to the resistance of nanopore 116 is reduced, the signal-to-noise ratio is not directly proportional to the resistance ratio. For example, in some embodiments, when the fluid resistance of channel 122 is about 10% of the resistance of nanopore 116, the signal-to-noise ratio is about 30% of its optimal value.

[0162] Channel 122 may be formed in a wafer, and after via 114 is formed therethrough, an oxide layer may be used to reduce the diameter of via 114 through base layer 112, thereby enabling the amount of oxidation to adjust the aspect ratio.

[0163] A sensor electrode 126 or sensor element is disposed between the nanopore 116 and at least a portion of the channel 122. In this example, the sensor electrode 126 forms an electrical transduction element. More generally, the sensor electrode 126 may be adapted to form any of the various types of electrical transduction elements disclosed in WO 2016 / 127007.

[0164] The sensor electrode 126 is at least partially exposed to the passage 114 in the nanopore sensor 102 and is configured with a connection 128 for measuring the potential of the fluid at the location of the sensor electrode 126 when the fluid is provided in the passage 114. The sensor electrode 126, together with the nanopore layer 110 and the base layer 112, defines the walls of the passage 114. The connection 128 can be a wire bond to a separate electronic circuit 130 that is configured to analyze the signal obtained from the sensor electrode 126.

[0165] Analyte reservoir 106 can serve as a first fluid reservoir, while outlet chamber 108 can serve as a second fluid reservoir. Structure 100 can at least partially separate analyte reservoir 106 from outlet chamber 108, and passageway 114 of sensor 102 connects analyte reservoir 106 to outlet chamber 108.

[0166] In use, the passage 114 of each nanopore sensor 102 is occupied by fluid. Further, the drive electrodes 132 in the analyte reservoir 106 and the outlet chamber 108 include at least one corresponding cis electrode 132a (also referred to as the analyte electrode) and at least one corresponding trans electrode 132b (also referred to as the outlet electrode), the at least one corresponding cis electrode and the at least one corresponding trans electrode being configured to impose a potential difference across the passage 114 of the nanopore sensors 102 in the array 104 between the analyte reservoir 106 and the outlet chamber 108.

[0167] 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 arranged to face the analyte reservoir 106 and define a cis plane 136 and a trans surface 138 of the base layer 112 arranged to face the outlet chamber 108 and define a trans plane 140. The cis plane 136 and the trans plane 140 are formed by Figure 2 、 3(b) 7( b). Sensor electrode 126 is embedded within structure 100 between cis plane 136 and trans plane 140. Nanopore 116 may be substantially located on cis plane 136 at first end 120 of passage 114, while second end 124 of passage 114 may be substantially located on trans plane 140.

[0168] like Figure 2 As shown in the assembly of FIG. 1 , the sensor electrodes 126 can be at least partially embedded in the structure 100 between the nanoporous layer 110 and the base layer 112. In other words, the sensor electrodes 126 are sandwiched or laminated between the nanoporous layer 110 and the base layer 112.

[0169] The nanoporous layer 110 has pores 142 formed at the first end 120 of the via 114. Figure 2 In the example of, nanopore 116 is configured at the first end 120 of passage 114, on one side of hole 142, substantially on cis plane 136. As shown, sensor electrode 126 is configured on the side of hole 142 opposite to nanopore 116. Hole 142 is shown as a cup-shaped recess, and membrane 118 is shown across the edge of the hole in cross section. Hole 142 is configured to receive analyte that has passed through nanopore 116. Note that hole 142 is connected to channel 122 by hole orifice 142a fluid, and the hole orifice can be described as hole outlet. Hole orifice 142a plays the role of enabling the analyte chamber to be fluidically connected to the outlet chamber. Hole orifice 142a is not used as nanopore. In some embodiments, hole orifice 142a is configured to enable analyte to pass through it unimpeded (i.e., without affecting the movement of analyte from analyte reservoir 106 to outlet chamber 108).

[0170] Although the pore orifice 142a provides a fluid connection between the analyte reservoir 106 and the outlet chamber 108, the analyte that has passed through the nanopore 116 can be retained in the pore 142. The pore 142 and the channel 122 can be considered part of the outlet chamber 108. Figure 2 In the example shown in , the hole aperture 142a is positioned at the center of the base of the hole 142 within the sensor electrode 126 .

[0171] The pores 142, and more generally the nanoporous layer 110, are configured to support a membrane 118, such as a polymer membrane or a lipid bilayer, which can be referred to as a fluidic membrane. The nanoporous layer 110 can be fabricated separately from the base layer 112. The nanoporous layer 110 can be formed from a material different from that of the base layer 112. The nanoporous layer 110 can be formed from at least one of: a photolithographically prepared material; a molded polymer; or a laser-etched plastic.

[0172] In some embodiments, the sensor electrode 126 can be directly connected to the base or gate of a sensing transistor 153 (as shown in FIG5( a) and described further below) to measure the potential of the fluid at the location of the sensor electrode 126 when the fluid is provided in the passage 114. In some cases, the sensor electrode 126 can be connected to an edge connector or optionally bonded to the electronic circuit 130 via conductive vias and / or interconnecting leads, which is an example of measurement circuitry outside the structure. The sensing transistor 153 can be a field effect transistor (FET), and the configuration of the sensing transistor 153 and its optional integration into the structure 100 are described in the examples below. In some embodiments, the sensing transistor 153 (not shown) can be located in the electronic circuit 130.

[0173] Figure 2 The nanopore sensor 102 shown in FIG is an example in which the sensor electrode 126 may be formed on the base layer 112. Figure 2 The sensor electrodes 126 can be formed directly on the base layer 112, but alternatively, they can be formed separately on the sensing layer 144, as depicted in Figures 3(a) and 3(b). After the sensor electrodes 126 are formed on the sensing layer 144, the sensing layer 144 can then be sandwiched between the nanoporous layer 110 and the base layer 112, resulting in the structure shown in Figure 3(a).

[0174] The sensing layer 144 can be manufactured in a similar manner to the base layer 112, wherein the wafer has passages 114 formed therethrough that are 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 the sensing layer 144, surrounding the passages 114 on one side of the sensing layer 144. The passages 114 passing through the sensing layer 144 and the sensor electrodes 126 formed around these passages 114 are arranged to have a spacing or layout that results in alignment with the channels 122 on the base layer 112. When fixed together, the passages 114 of the sensing layer 144 are aligned with the channels 122 of the base layer 112.

[0175] For example, the nanopore layer 110 may be made of a polymer, which may be molded or photolithographically etched; the base layer 112 may be formed from a silicon wafer; and / or the sensing layer 144 may be a CMOS wafer.

[0176] Sensing layer 144 can be aligned and bonded to base layer 112 in one of two orientations. In one orientation (not shown), sensor electrode 126 remains fully exposed after bonding, that is, sensor electrode 126 is: not sandwiched between sensing layer 144; located distally from base layer 112 after sensing layer 144 is secured to base layer 112; and subsequently sandwiched between sensing layer 144 and nanopore layer 110. In another orientation, as shown in FIG3( a ), sensor electrode 126 is formed on top of sensing layer 144, which is then inverted before bonding to base layer 112, such that sensor electrode 126 faces downward (as viewed) and is sandwiched between sensing layer 144 and base layer 112. Prior to bonding in this configuration, the section of the oxide layer on base layer 112 surrounding channel 122 can be etched or otherwise removed to create cavity 146, thereby increasing the area of ​​sensor electrode 126 exposed to via 114 after bonding. The area of ​​the exposed sensor electrode 126 may be maximized to increase contact with the fluid in the passageway 114 .

[0177] The pores 142 of the nanoporous layer 110 align with the vias 114 and the sensor electrodes 126 and are bonded to the sensing layer 144, with the sensor electrodes 126 sandwiched between them. Referring to FIG3( b ), it is noted that the sensing layer 144 is fabricated from the bottom up, with the final stage being the application of the sensor electrodes 126 on top. During assembly, the sensing layer 144 is flipped over so that the sensor electrodes 126 on top now face downward, as shown in FIG3( b ). Etching away the cavities 146 in the oxide layer (gray) of the base layer 112 means that the sensor electrodes 126 are fully exposed.

[0178] The sensor electrode 126 remains at least partially exposed to the passage 114 and is configured with a connection for measuring the potential of the fluid at the location of the sensor electrode 126 at the nanopore 116 when the fluid is provided in the passage 114. The arrangement of the sensor electrode 126 (which minimizes the surface area of ​​the sensor electrode openly facing one of the analyte reservoir 106 or the outlet chamber 108 (e.g., Figure 2 3( a)) is used to limit exposure to the analyte reservoir 106 or the outlet chamber 108 to prevent contamination of the surface of the sensor electrode 126. One such example is shown in FIG3( a), which shows the sensor electrode 126 substantially partially enclosed in the passage 114. Before being filled with a fluid, or during formation of the amphiphilic membrane supporting the bionanopore, the surface of the sensor electrode 126 may be exposed to a fluid that may contaminate the surface of the sensor electrode 126, so if there is a risk of contamination, it can be mitigated.

[0179] In one configuration, at least a portion of the sensor electrode 126 can be arranged to face away from the pore 142 toward the channel 122, as shown in FIG3( a). The exposed portion of the sensor electrode 126 provides a connection to the fluid in the passage 114 to sense fluctuations in the fluid potential at the sensor electrode 126 when 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) to connect to an electronic circuit 130 (such as a measurement circuit or a control circuit), which can be separate from the structure 100, as shown in FIG3( a).

[0180] In each of the examples, sensor electrode 126 can be configured in various configurations to be exposed to the fluid within passageway 114, and can be at least one of: at least partially covering the wall of passageway 114; covering a portion of the wall of passageway 114 in cross-section; forming a ring around passageway 114; forming a surface of base layer 112 or sensing layer 144 at least partially surrounding passageway 114 and having an exposed portion disposed facing analyte reservoir 106; forming a surface of sensing layer 144 at least partially surrounding passageway 114 and having an exposed portion disposed facing outlet chamber 108. Specifically, cavity 146 can be formed in passageway 114 to create an area that increases the exposed area of ​​sensor electrode 126 and allows for contact with an increased amount of fluid. Cavity 146 is formed by a recess formed in base layer 112 and / or sensing layer 144 before base layer 112 and sensing layer 144 are aligned and connected.

[0181] While the sensor electrodes 126 may be minimally exposed to the fluid in the pore, such as in the form of nanowires, the inventors have developed the examples herein to optimize the performance and improve manufacturability of the nanopore sensor 102 .

[0182] like Figure 2 and 3(a) , the sensor electrode 126 is substantially planar and shaped to accommodate the passage 114. In other words, the sensor electrode 126 is configured to enable unimpeded fluid communication between the analyte reservoir 106 and the outlet chamber 108, which may be achieved by: (i) shaping the sensor electrode 126 to extend around the passage 114 or the orifice 142a and / or (ii) forming the sensor orifice 148 in the sensor electrode 126.

[0183] The footprint of the exposed portion of sensor electrode 126 may be any shape. Figure 2The hole 142 and the cavity 146 of FIG. 3( a ) can be cylindrical, such that the floor of the hole 142 is circular, or the planar surface of the cavity 146 is curved. These configurations result in the exposed portion of the sensor electrode 126 being circular or disc-shaped. In the example shown, the sensor aperture 148 is provided such that the footprint of the exposed portion is shaped like a ring. The exposed area of ​​the sensor electrode 126 can be maximized, which can mean covering at least one face or surface of the hole 142 and / or cavity 146.

[0184] The sensor electrode 126 and the sensor orifice 148 are shown as being circular, but 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 orifice 148 may be in the range of 100:1 to 10:1, or about 2:1. In the case of a non-circular shape, the ratio of the square root of the area may have the same value.

[0185] Alternatively, the area of ​​the exposed portion of the sensor electrode 126 may be expressed as a ratio to the as-seen area or footprint of the sensor aperture 148 , which may be about 1:1, or about 10:1, or about 100:1.

[0186] For example, the diameter (or maximum dimension) of the sensor electrode 126 can range from 10 μm to 50 μm, and the diameter (or maximum dimension) of the sensor orifice 148 can range from 0.5 μm or greater. The sensor orifice 148 does not function as a sensor, so its size has no upper limit within the bounds of minimizing the constraints of the remaining area of ​​the sensor electrode 126.

[0187] The sensor electrodes 126 may be formed from a suitable conductive material, such as platinum or gold.

[0188] Although Figure 2 and 3(a) The structure 100 and array 104 have sensor electrodes 126 with connections 128 to separate electronic circuits 130, but FIG3( b ) illustrates that the structure 100 and array 104 can accommodate an integrated circuit 150. The integrated circuit 150 can incorporate one or more of the functions of the electronic circuit 130. In other words, various functions that can be implemented on separate electronic circuits 130 (such as sensing, amplification, control, filtering, readout, etc.) can alternatively be implemented on the integrated circuit 150. The integrated circuit 150 can be formed on a separate layer or wafer and then connected to the sensing layer 144 having the sensor electrodes 126 thereon. However, the inventors contemplate that the integrated circuit 150 is fabricated within the sensing layer 144 along with the sensor electrodes 126. An integrated circuit 150 can be provided for each nanopore sensor 102.

[0189] In some methods, after fabricating the sensing layer 144 having the integrated circuit 150 and sensor electrodes 126 formed and exposed on one side, the sensing structure is flipped over and bonded to the base layer in the same manner as with respect to FIG. 3( a). Connection 128 (not shown in FIG. 3( b)) connects the integrated circuit 150 to a connector 151 to transmit signals or data generated by the integrated circuit 150 to the outside of the structure. Connection 128 can be connected to connector 151, as shown in FIG. 4( e), although other configurations are also possible. In the case where the sensing layer 144 is connected to the base layer 112, the nanoporous layer 110 can be formed thereon so that the sensing layer 144 is sandwiched between the nanoporous layer 110 and the base layer 112. As before FIG. 3( a), when bonded together, the vias of the sensing layer 144 align with the channels of the base layer 112, and the pores 142 of the nanoporous layer 142 form part of the vias 114.

[0190] In use, the electronic circuit 130 and / or the integrated circuit 150 are configured to detect a change in resistance at the nanopore 116 when an analyte (e.g., a polymer) passes through the nanopore 116. The change in resistance is detected by the fluid in the nanopore sensor 102 (strictly speaking, a measure of resistance is detected as a voltage across an effective voltage divider as described above). In the array 104, the integrated circuit 150 of each nanopore sensor 102 can be communicatively addressable. Due to parasitics, communication noise, and background noise, the detected resistance can be difficult to read directly using an off-board processor. To provide a better signal (i.e., a cleaner signal with reduced noise) to the processor, the integrated circuit 150 can be configured to locally transform, modify, or otherwise process the signal resulting from the detection of the polynucleotide or other analyte passing through the nanopore 116. The integrated circuit 150 can be configured to perform at least one of the following: amplify the signal, such as to amplify the voltage level of the signal; filter the signal, for example, to remove noise; sample the signal; or digitize the signal using an analog-to-digital converter (ADC) implemented in the electronic circuit 130.

[0191] The integrated circuit 150 may be formed within the footprint of the nanopore sensor 102 within the array 104 of the structure 100 .

[0192] For example, each nanopore sensor 102 of the array 104 can be contained within a pixel 101, which serves as the footprint of the nanopore sensor 102, as seen in FIG4(a), which can be considered to represent the schematic plan view of the nanopore sensor 102 shown in FIG3(b). As 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 can be arranged to prevent noise interference generated by the integrated circuit 150 from being detected by the sensor electrode 126. For example, the integrated circuit 150 can be separated from the nanopore sensor 102, as depicted in FIG4(a). This separation can be implemented by configuring the integrated circuit 150 to be located outside the pixel 101, as seen.

[0193] This separation can also simplify the manufacturing process. Alternatively, the integrated circuit 150 can be spaced apart from the sensor electrodes 126 (i.e., the distance between the sensor electrodes 126 and the integrated circuit 150 in the depth direction or the thickness of the structure 100 is increased) to minimize noise interference. Note that the depth direction of Figure 4(a) is the direction into and out of the page, as viewed.

[0194] In the example shown, the pixels 101 are square and have a side length of 20 μm, but in other examples the side length may range from 10 μm to 50 μm.

[0195] 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.

[0196] Other arrangements are contemplated. In some embodiments, the sensor electrodes 126 can be larger than the example shown in FIG. 4( a ), for example, covering substantially all of the area of ​​the nanopore sensor 102 . In some embodiments, the sensor electrodes 126 can have other shapes that cover more area, such as square or rectangular. The size of the sensor electrodes 126 can be up to 50 μm, in which case their area can be up to 250 μm 2 , depending on its shape.

[0197] For packing efficiency, the pixels 101 may be in a tessellated shape, and for example, the tessellated shape may be a hexagon.

[0198] Each nanopore sensor 102 has a channel 114, although during the fabrication of the base layer 112, more channels 122 than necessary may be created in the base layer 112, depending on the fabrication method. Some fabrication methods (e.g., reactive ion etching) can etch a single channel 122 for each pixel 101. Other methods (e.g., light-assisted electrochemical etching) require etching a high-density array of channels 122 simultaneously to maintain the geometry of these channels 122. In this case, unused channels 122 in the base layer 112 are blocked during fabrication of the array 104, so that each pixel 101 is provided with only one channel 122 and one channel 114. The density of channels 122 formed in the base layer 112 can vary. For comparison, FIG4(b) shows a cross-section of a nanopore sensor 102 having a lower density of blocked channels 122a than that shown in FIG4(a). 4( b ), the channel 122 may be blocked before the sensing layer 144 is added to the base layer 112, or may be blocked by the substrate of the sensing layer 144. It should be noted that FIG4( b ) shows a portion of two nanopore sensors 102, each with its own passage 114, and the nanopore layer 110 has not yet been added to the sensing layer 144.

[0199] FIG4(c) shows the pixels 101 of the nanopore sensor 102 of FIG4(a) arranged in a 6×6 layout providing an array 104 of 36 nanopore sensors 102, while FIG4(d) shows an 18×18 array having 324 nanopore sensors 102. The array size can be 1000×1000, providing 1,000,000 nanopore sensors 102. In this example, the footprint of an array of one million nanopore sensors 102 of the type shown in FIG4(a) would be 4 cm 2 However, nanopore sensors 102 with pixels 101 as small as 5 μm can reduce the footprint of a one million nanopore sensor array 104 to 25 mm 2 The array size may be 100,000. The array 104 may include any number of nanopore sensors 102, between 1,000 and 10 million nanopore sensors 102.

[0200] Figure 4 (e) shows an array 104 of nanopore sensors 102 arranged in a structure 100 provided in a device 149 as described herein to accommodate and analyze polymer analytes (such as nucleic acids). Device 149 may also be referred to as a sensor device or measurement system. Array 104 may be a subassembly of device 149. Array 104 may be a disposable component and replaceable. Additionally or alternatively, the nanopore layer 110 of array 104 may be a disposable component and replaceable. Although some aspects of the present invention generally relate to device 149, other aspects of the present invention may also relate to nanopore sensors 102 or nanopore sensors 102 having an array of nanopore structures 104. Device 149 may include an electronic circuit 130 as described above.

[0201] 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 prior to further analysis by electronic circuitry 130 of device 149.

[0202] In some embodiments, the device 149 houses the structure 100 to separate and define the analyte reservoir 106 and the outlet chamber 108. Although often referred to as cis and trans, respectively, analytes 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 passage 114 therethrough to fluidically connect the analyte reservoir 106 and the outlet chamber 108. For example, drive electrodes 132 in the analyte reservoir 106 and the outlet chamber 108 can impose a potential difference across the passage 114 between the analyte reservoir 106 and the outlet chamber 108 to induce the analyte to flow from the analyte reservoir 106 to the outlet chamber 108. The drive electrodes 132 can be configured such that the potential difference across all nanopore sensors 102 is substantially the same.

[0203] Additionally or alternatively, the device 149 may be configured to use other techniques to induce analyte from the analyte reservoir 106 to the outlet chamber 108. As the analyte passes through the nanopore 116, the fluctuations in electrical potential due to changes in ionic current are detected by the sensor electrode 126.

[0204] The sensor electrode 126 can serve as or be directly connected to the base or gate of a sensing transistor 153 (as shown in FIG5( 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 voltage at the drain can be read out 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.

[0205] In the above example, each pixel 101 has its own integrated circuit 150, but the integrated circuit 150 can be configured to serve multiple nanopore sensors 102. In Figure 4(f), four nanopore sensors 102 are shown as a sensor module 102a (which can be part of a larger array of nanopore sensors 102), where the integrated circuit 150 is common to the four centrally located sensor electrodes 126, as shown. Other configurations are possible. In such a module configuration, information or data obtained from each individual nanopore sensor 102 is addressable for control and / or retrieval. While the above example has a dedicated integrated circuit 150 for each nanopore sensor 102, combining the nanopore sensors 102 in the sensor module 102a allows for improved layout efficiency. For example, because a common filter is used for each of the nanopores 116 within the sensor module 102a, this efficiency improvement can be achieved. This is possible if the integrated circuit 150 is sequentially switched or multiplexed between the individual nanopore sensors 102. By sharing functionality among nanopore sensors 102, the footprint of integrated circuit 150 may be reduced, or alternatively, more functionality may be accommodated.

[0206] FIG5 (a) is a schematic representation of the direct connection of the sensor electrode 126 to each nanopore sensor 102 in the array 104. The cis electrode 132a can be connected to ground, while the shift voltage is applied to the counter electrode 132b. The resistance of the nanopore 116 and the resistance of the channel 122 (which is configured to act as a fluid resistor) dominate the circuit between the cis electrode 132a and the counter electrode 132b through each passage 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 fluid resistor are approximately the same, so that the electrode positioned therebetween is optimally positioned to detect changes in the nanopore resistance caused by the analyte passing therethrough. As described above, the sensor electrode 126 resides 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 the nanopore 116 and channel 122 is much greater than the bulk fluid resistance of the analyte reservoir 106 and outlet chamber 108, which means that Figure 5(a) can be used to simulate the circuit between the cis electrode 132a and the counter electrode 132b.

[0207] The circuit system includes a sensing circuit 152 that measures the fluid potential at the sensor electrode 126 of the nanopore sensor 102 to obtain measurements from the nanopore 116. The sensing circuit 152 can include a sensing transistor 153, which can be, for example, a field effect transistor (FET). In this case, the sensor electrode 126 can be connected to the base of the sensing transistor 153. The sensing circuit 152 can reside at least partially in the integrated circuit 150. Thus, the sensor electrode 126 can be connected to a sensor terminal 154 of the sensing circuit 152, as shown in FIG5(a).

[0208] Optionally, the sensing circuit 152 may include a control circuit 155 that applies a signal to the sensor electrode 126 to change the potential difference imposed across the nanopore 116 by the drive electrode 132 in response to the control signal. The control circuit 155 may include a control terminal 156, which may be, for example, a field effect transistor (FET). In this case, the sensor electrode 126 may be connected to the drain of the control terminal 156. The control circuit 155 may reside at least partially in the integrated circuit 150. Thus, the sensor electrode 126 may be connected to the control terminal 156 of the control circuit 155 for applying the control signal, as shown in FIG5( a).

[0209] Application of a control signal can change the potential difference imposed across an individual nanopore 116 by changing 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 (e.g., species) to change the direction of its movement through the pathway 114.

[0210] In some cases, the applied voltage may be an alternating voltage, although other voltage waveforms (eg, ramp, step, pulse, DC) may alternatively be applied.

[0211] 5( a) enables common electrodes to be configured for each of the analyte reservoirs 106 and outlet chambers 108, and each nanopore sensor 102 can be operated to detect a disturbance in the ionic current across the passage 114 by detecting a change in electrical potential caused by a change in nanopore resistance. Furthermore, the integrated circuit 150 enables each nanopore sensor 102 within the array 104 to be individually controlled, such that the sensor electrode 126 can detect analyte passing through the nanopore 116, for example, by connection to a sensing transistor 153, or control the flow of charged analytes (e.g., species) through the passage 114 of an individual nanopore sensor 102 in the array 104 by adjusting the voltage applied to the sensor electrode 126 using, for example, a control transistor 156. Controlling the flow of charged analytes (e.g., species) through the passage 114 of an individual nanopore sensor 102 in the array 104 allows the analyte to pass through the nanopore 116 or analytes that block the nanopore 116 to pass back or be ejected by the voltage applied by the control transistor 156. This action can be described as a "flick" or "rejection" and occurs by using a control voltage so that the analyte's passage through the pathway 114 from one side of the structure 100 is modified (stopped, reversed, or accelerated). The control voltage can be applied individually to each nanopore 116 because each nanopore sensor 102 is individually addressable for control and sensing. Specifically, applying a control signal to the sensor electrode 126 in each nanopore sensor 102 means that the voltage near the nanopore 116 at each pixel 101 can be controlled.

[0212] The 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 circumstances: upon detection of a blocked nanopore 116; when the detected analyte is no longer of interest or is to be ejected for the purpose of enabling another sample to be received and measured; and to alter the rate at which the analyte is induced to enter or leave the nanopore 116.

[0213] Electronic sensors inevitably have capacitance, resistance, and inductance associated with the path along which the sensor signal travels, which can be referred to as parasitics. This is due to the properties of the materials from which the sensor is constructed, its geometry, and the available methods for manufacturing it. Without any type 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 will limit the bandwidth to 1 / (2πRC).

[0214] FIG5(b) is an alternative schematic diagram to FIG5(a) showing a resistor model 161 of the nanopore 116 and channel 122 and further comprising a compensation circuit 160 connected to a voltage divider. According to some embodiments, the compensation circuit 160 has an in-line amplifier 168 with a gain of G connected to the output of the sensor electrode 126, which is affected by the 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, which has a feedback amplifier 170 with a gain of H and a compensation capacitor C representing the compensation capacitance. 补偿 capacitor C.

[0215] Parasitic input capacitance 162 is shown arranged in parallel with the resistor representing channel 122. The parasitic input capacitance represents the parasitic capacitance of at least one of the following: membrane 118 in which nanopore 116 is located; the fluid walls of channel 122; sensor electrode 126; and trace capacitance associated with the connector or wire bond. Sensor electrode 126 is effectively connected to the midpoint of the voltage divider between nanopore 116 and channel 122 and to compensation circuit 160. The connection to the reverse or flick voltage is represented by a flick switch 164, such as a FET. An optional protection switch 166 is shown implemented between sensor electrode 126 and compensation circuit 160. This protection switch 166, which may be implemented using a FET, can serve to isolate compensation circuit 160 and / or any sensing circuitry connected thereto from the flick voltage applied via flick switch 164.

[0216] In general, the compensation circuit 160 mitigates the effect of the total parasitic input capacitance 162 at the input to the sensing circuit 152. Although the parasitic capacitance resides in various elements of the nanopore sensor 102, it can be modeled as shown in FIG5(b). Without being limited by a particular theory, the total parasitic input capacitance 162C p can be considered as the sum of parasitic capacitances as follows.

[0217] C p =C 膜 +C 流体壁 +C 电极 +C 迹线

[0218] The rate at which the input capacitor charges is proportional to the current flowing through it. Furthermore, the resistor limits the charging current to a finite value. Compensation circuit 160 serves to provide additional current to charge the input capacitor more quickly, thereby increasing bandwidth.

[0219] The compensation circuit 160 of FIG5(b) has an in-line amplifier 168 with a gain of G connected to the output of the sensor electrode 126, which is affected by the parasitic input capacitance 162. The output of the in-line amplifier 168 has a feedback loop connected to its input, which has a feedback amplifier 170 with a gain of H and a compensation capacitor C 补偿 Compensation capacitor C172.

[0220] In some embodiments, the input voltage is amplified and fed back through 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:

[0221] C 有效 =C in -C 补偿

[0222] in

[0223] C 补偿 =C×(G×H–1)

[0224] The components of the compensation circuit 160 are configured so that the total parasitic input capacitance C p 162 is substantially canceled or offset. In practice, the degree of compensation is limited by the dynamic changes in component values ​​and parameters (e.g., temperature dependencies). If the capacitance C, the in-line gain G, or the feedback gain H are made adjustable, the compensation circuit 160 can compensate for a range of different parasitic input capacitance values, so the feedback amplifier 170 is shown as variable in FIG5( b). The gain G can be fixed so that the output from the compensation circuit 160 has a consistent gain, so the compensation capacitor C 172 and / or the feedback gain H can be varied.

[0225] The front-end electronics may reside at least partially in an integrated circuit 150, which is graphically represented in FIG5( 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 influence the movement of the analyte within the nanopore 116, such as by flicking, by applying a reverse voltage, and by amplifying the signal from the nanopore sensor 102. The integrated circuit 150 or electronic circuitry 130 may additionally incorporate further processing of the signal (e.g., filtering), and in the case of the integrated circuit 150, may include circuitry for storing information locally in the sensor 102 for managed communication with an external processor.

[0226] Each nanopore sensor 102, such as Figure 2 3(a) and those nanopore sensors shown in FIG. 3(c) can be addressable. FIG. 5(c) shows the nanopore sensor 102 of FIG. 3(b) incorporating an integrated circuit 150 within the pixel 101 shown in FIG. 4(a) and also addressable via row select and column buses. FIG. 5(d) is an example of row-column readout circuitry 174 connected to each nanopore sensor 102 in an array 104 (such as the array 104 shown in FIG. 4(d)) via the row select and column bus connections shown in FIG. 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 circuitry 174 can be connected to the integrated circuit 150 of each nanopore sensor 102 or a group of nanopore sensors 102, but can be directly connected to the sensor electrodes 126 in each nanopore sensor 102 within the array 104.

[0227] The above example describes an option in which the sensor electrode 126 can be connected to the integrated circuit 150 and has dual functionality when a control voltage is applied (i.e., the sensor electrode 126 can be used to sense changes in ion flow when an analyte passes through the nanopore 116 and, under the control of the control circuit 155, generate a potential within the pathway 114 and generate a potential difference across the pathway 114 between the cis electrode 132a and / or the trans electrode 132b). In this case, the sensor electrode 126 is directly connected to the control terminal 156, which is a terminal of the integrated circuit 150, to generate a potential within the pathway 114, as shown in FIG5( a) and described further below.

[0228] In some embodiments, the sensing and control functions in each nanopore sensor 102 may be implemented by separate electrodes, for example as follows. FIG6( a ) shows the sensor electrodes 126 and control electrodes 182 arranged like a ring, while FIG6( a ) shows the sensor electrodes 126 and control electrodes 182 arranged like a ring. Figures 6(b) to 6(e)1 is a schematic cross-sectional view of the nanopore sensor 102 having a configuration in which a control electrode 182 is provided in addition to the sensor electrode 126. In this case, the control electrode 182 is connected to the control terminal 156 of the control circuit 155 to generate a potential within the passage 114.

[0229] In the examples herein, the sensor electrodes 126 have been described as rings, as illustrated in FIG4( a). The sensor electrodes 126 may also be implemented as exposed wires. The sensor electrodes 126 may be nanowires, but may occupy a larger surface area, such as substantially the entire base of the hole 142 (as shown in FIG6( e) ) or one face of the recess 146. Similarly, the separate control electrode 182 may be a nanowire, but may have a large surface area, as shown in FIG6( d).

[0230] From a manufacturability and cost perspective, a basic embodiment of the control electrode 182 is shown in FIG6( a ), wherein a substantially annular footprint that fits within the base of the well 142 is maintained, with half of the footprint forming the sensor electrode 126, and the other half, physically disconnected or decoupled from the sensor side, forming the control electrode 182. There are no wired or solid-state 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 semicircular shapes of equal size occupying the footprint. The size of the electrodes can vary, and for example, the surface area of ​​the control electrode 182 can be larger than that of the sensor electrode 126 to increase conductivity with the fluid within the passageway 114.

[0231] Having separate sensor and control electrodes 126, 182 can simplify the integrated circuit 150 because, by being separate, an additional degree of isolation is provided, although they will still be connected by the fluid in the passage 114. However, the need for isolation switches to protect, for example, the compensation circuit 160, which may form part of the sensing circuit 152, from voltages applied by the control circuit 155 can be avoided. The sensor and control electrodes 126, 182 can be customized in shape, size, and configuration to be optimized for their purpose.

[0232] FIG6( b ) illustrates how the sensor electrode 126 of FIG3( b ) can be separated into separate sensor electrodes 126 and control electrodes 182. In this example, the sensor and control electrodes 126, 182 extend in the same plane. In an alternative configuration shown in FIG6( c ), the sensor electrode 126 resides in the cavity 146 and extends in a plane parallel to the cis- and trans-surfaces 134, 138, while the control electrode 182 extends in the channel 122 and perpendicularly from the cis- and trans-surfaces 134, 138. In FIG6( c ), the sensor electrode 126 is shaped like a ring, while the control electrode 182 is shaped like a cylinder. In yet another alternative, as shown in FIG6( d ), the sensor electrode 126 resides in the cavity 146 and extends in a plane parallel to the cis- and trans-surfaces 134, 138, while the control electrode 182 extends in the channel 122 and the cavity 146, thereby extending in both vertical and horizontal planes, as seen. FIG6(e), similar to FIG6(b), shows sensor and control electrodes 126, 182 formed at the base of the aperture 142, which may provide easier manufacturing.

[0233] As described above, electronic sensors inevitably have capacitance, resistance, and inductance associated with the path along which the sensor signal travels, which can be referred to as parasitic phenomena, including parasitic capacitance. In addition to or in lieu of the compensation circuit 160 described above, the array 104 and the nanopore sensor 102 therein can be manufactured with a guard conductor 184 incorporated therein, as shown in Figures 7(b) to (d), while Figure 7(a) shows a first schematic circuit 201 and a second schematic circuit 202 with and without the guard conductor 184 to illustrate how the guard conductor 184 is configured.

[0234] In the first schematic circuit 201 on the left hand side of FIG. 7( a ), the parasitic capacitance C 寄生 is 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 (eg, the sensor electrode 126) may carry a voltage V 传感器 , and the second conductive element 204 (eg, a conductive substrate) can carry a different voltage V 衬底 .

[0235] The protection is shown as being implemented in the second schematic circuit 202 on the right hand side, wherein the guard conductor 184 as the third conductive element is arranged between the first conductive element 203 and the second conductive element 204 carrying the signal, so that the two parasitic capacitances C par1 、C par2In this second schematic circuit 202, parasitic capacitance occurs between (i) the first conductive element 203 carrying the signal and the guard conductor 184 (ie, C par1 ) and (ii) between the guard conductor 184 and the second conductive element 204 (ie, 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. Therefore, the parasitic capacitance C across the first conductive element 203 and the guard conductor 184 is par1 There is no voltage difference.

[0236] For a capacitor, the current is given by:

[0237] In the diagram on the right, V 保护 =V 传感器 ,therefore

[0238] No current flows through capacitor C par1 , so the effective capacitance is zero. The capacitance between the guard conductor 184 and the substrate conductor must still charge, but the buffer 205 can source much more current than the high impedance sensor input, so it charges much faster.

[0239] When V 保护 Follow V exactly 传感器 These conditions are met when , depending on the performance of the buffer 205 having sufficient bandwidth to enable the capacitance to be ineffective. A precise buffer 205 with a bandwidth of several MHz can be implemented.

[0240] Figure 7(b) is similar to Figure 2(b) and by comparison, guard conductor 184 is shown extending vertically along the length of channel 122 between oxide layer 192 as seen, and continuing horizontally along the top of base layer 112 below sensor electrode 126 as seen. Notably, both sensor electrode 126 and guard conductor 184 are connected to separate electronic circuitry 130. In this configuration, guard conductor 184 blocks current flow in the parasitic capacitance between sensor electrode 126 and the substrate of base layer 112. The conductive guard may at least partially comprise guard conductor 184 and an insulating layer, such as oxide layer 192, that insulates guard conductor 184 from the conductor being protected or from which it is being protected. The insulating layer is not part of guard conductor 184 and serves to isolate guard conductor 184 from surrounding conductors. Thus, the insulating layer may be a non-conductive component of structure 100. Guard conductor 184 may be a conductor inserted into the middle of the parasitic capacitance to bisect it, which is possible because capacitors are inherently insulators, and thus guard conductor 184 is positioned within the existing insulating layer.

[0241] The conductive guard (including the insulating layer) can be configured in a variety of different configurations or combinations thereof, including at least one of: extending over at least a portion of the nanopore layer 110 to separate the nanopore layer 110 from the analyte in the analyte reservoir 106, as shown in Figure 7(c), which protects the solution below the nanopore 116 from the solution above; extending between at least a portion of the nanopore layer 110 and the sensing layer 144 to separate the sensor electrode 126 and the integrated circuit 150 from the solution in the channel 106, also as shown in Figure 7(c); extending between the base layer 112 and the sensing layer 144 to at least partially separate the sensor electrode 126 and the integrated circuit 150 from the base layer 112, also as shown in Figure 7(c); and multiple conductive guards, as shown in Figure 7(d), wherein 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 sensing layer 144 and the base layer 112.

[0242] Based on the teachings herein, skilled artisans will appreciate that one or a combination of the guard arrangements taught herein may be implemented. It should also be understood that guard conductor 184 may be disposed in a nanopore structure array 104 (eg, array 104 of FIG. 4( c )).

[0243] It should be noted that Figures 7(a) to 7(d)The advantage of the guard-based capacitance compensation technique shown in is that it generally does not significantly increase the noise level of the signal. However, when the potential difference across the membrane 118 is used to drive the analyte being studied through the nanopore 116, this technique cannot compensate for the membrane capacitance, but can drive the analyte in another way (e.g., pressure). On the other hand, the compensation circuit 160 can compensate for the entire input capacitance, but does so at the expense of increased noise. The noise gain of the compensation capacitor 172 increases with frequency. Therefore, the noise in the input signal is scaled by this feedback gain "G" and added to the overall noise. This becomes important at higher frequencies or when compensating for larger input capacitances. The guard conductors 184 shown in Figures 7(b) to (d) can be implemented in the array 104 in any combination and / or in combination with the compensation circuit 160.

[0244] The nanopore sensor 102 can be manufactured using a variety of different techniques, and for example, see Figure 2 (which indicates other sensors taught in this application) functionality is taught. Although Figure 2 Only one of the nanopore sensors 102 of the array 104 is shown, but the 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. Figure 2 Only one channel 122 is shown, formed through a Si wafer extending substantially perpendicular to the surface of the wafer. In practice, 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 desired, the diameter of channel 122 can be adjusted using techniques such as thermal oxidation to calibrate the aspect ratio. For example, the Si wafer and channels can be embedded in an oxide layer, which can be formed on a silicon wafer.

[0245] Figure 2 The example of FIG4 schematically shows a portion of a structure 100 having an array 104 of nanopore sensors 102 (only one of which is shown) and configured to separate an analyte reservoir 106 having a drive electrode 132 therein from an outlet chamber 108. All of the nanopore sensors 102 herein can be positioned in the structure 100 as shown in FIG4(e). The nanopore 116 is located in a fluid-filled passage 114 between the analyte reservoir 106 and the outlet chamber 108. The passage 114 is fluid-filled so that the analyte reservoir 106 and the outlet chamber 108 are fluidically connected. Specifically, the nanopore 116 is located in the fluid communication path between the analyte reservoir 106 and the outlet chamber 108.

[0246] Figure 8 and9 Two further examples of devices 149 comprising structure 100 are shown. In each case, structure 100 takes the form shown in FIG3( a) or 3( b) comprising nanopore layer 110, sensing layer 144 and base layer 112, as described in detail above (although in each case it may be taken as Figure 2 100 in the form shown in FIG).

[0247] exist Figure 8 and 9 In each of the examples, the structure 100 separates the analyte reservoir 106 from the outlet chamber 108 and connects to the printed circuit board 210, but with different configurations as follows.

[0248] exist Figure 8 In the example of FIG, the analyte reservoir 106 and the outlet chamber 108 are each formed by a respective gasket 216, 218 that seals against the nanoporous layer 110 and the base layer 112, respectively. For example, by respective members extending across the gaskets 216, 218, the analyte reservoir 106 and the outlet chamber 108 can be as shown. Figure 8 The shown open or can be closed.

[0249] exist Figure 8 In the example of FIG. 1 , the printed circuit board 210 is mounted to the base layer 112 by a mechanical bond 212 (e.g., adhesive) on the side opposite the nanoporous layer 110. Thus, the printed circuit board 210 is positioned outside of the outlet chamber 108, as shown in FIG. Figure 8 The sensing layer 144 is connected to the printed circuit board 210 by wire bonds 214 or any other suitable electrical connection. The area of ​​the nanoporous layer 110 is smaller than the area of ​​the sensing layer 144 to provide space for the wire bonds 214.

[0250] exist Figure 9 In the example of FIG, the printed circuit board 210 is mounted to the sensing layer 144 via solder bump connections 222 (e.g., adhesive) on the same side as the nanoporous layer 110. Thus, the area of ​​the nanoporous layer 110 is smaller than the area of ​​the sensing layer 144 to provide space for the solder bump connections 222. The solder bump connections 222 provide mechanical and electrical connections between the printed circuit board 210 and the sensing layer 144.

[0251] exist Figure 9 In the example of , the analyte reservoir 106 and the outlet chamber 108 are each formed in a respective flow cell 224, 226 that can be made of any suitable material (eg, plastic). The flow cells 224, 226 allow fluid to flow into and out of the analyte reservoir 106 and the outlet chamber 108.

[0252] The flow cell 224 forming the analyte reservoir 106 is sealed to the printed circuit board 210 surrounding the analyte reservoir 106 by a gasket 228 , and the printed circuit board 210 is sealed to the edge of the nanoporous layer 110 surrounding the analyte reservoir 106 by a sealant 230 .

[0253] The flow cell 224 forming the outlet chamber 108 is sealed to the base layer 112 surrounding the outlet chamber 108 by a gasket 232 .

[0254] Figure 8 and 9 It may be modified in various ways, such as to provide sealing at other locations (eg, around the outer edge of the base layer 112) and by any suitable means.

[0255] The electrical model of the nanopore sensor 102 has been described above. More generally, Figure 2 A voltage source (not shown) applies a potential difference between a drive electrode 132 disposed in the analyte reservoir 106 and the outlet chamber 108. The drive electrode 132 imposes a potential across the passage 114 containing the nanopore 116 and the channel 122. The nanopore resistance and the channel resistance are significantly higher than the overall fluid resistance of the analyte reservoir 106 and the outlet chamber 108, and therefore, the nanopore 116 and the channel 122 are the dominant components in the equivalent circuit. Figure 2 As shown in FIG, the sensor electrode 126 is located between the nanopore 116 and the channel 122 so that it can sense the fluid potential at the electrical transduction element in the passage 114. In other words, the sensor electrode 126 can sense a signal indicating a local potential fluctuation in the passage 114. Figure 2 The configuration in FIG. 1 is an example, but the sensor electrode 126 can be positioned in the analyte reservoir 106 or the outlet chamber 108. The sensor electrode 126 can function as a 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 in the passage 114. The sensor electrode 126 can detect fluctuations in voltage when a species (e.g., a strand of DNA) is translocated through the nanopore 116.

[0256] The embodiments herein have described a device 149 having a single analyte reservoir 106 separated from a single outlet chamber 108 by structure 100. Alternative arrangements may be implemented and include 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, in accordance with the teachings herein.

[0257] The nanopore layer 110 can be formed separately with an array of pores 142 that can be formed in a variety of ways, one of which is a polymer layer formed by photolithographic patterning. The pores 142 in the nanopore layer 110 are then aligned with the channels 122 of the base layer 112 so that each nanopore sensor 102 has a passage 114 defined by the pores 142 and the channels 122. Compared to the nanopores 116 located in the membrane 118, Figure 2 The holes 142 shown in FIG. 1 are substantial. Figure 2 Nanopores 116 are biological nanopores in membrane 118 (e.g., an amphiphilic membrane). Alternatively, each nanopore 116 can be a solid-state nanopore located in a solid-state membrane. The solid-state membrane itself can be nanoporous layer 110. Further alternatively, nanopores 116 can be biological nanopores located in a solid-state membrane. Depending on the size of nanopore 116 relative to the width of channel 122, which has a larger diameter, it can be said that a hole 142 is formed below nanopore 116. Thus, in each of the alternative nanopore configurations, nanopore 116 defines a portion of passage 114.

[0258] According to the various aspects described herein, any membrane 118 can be used. Suitable membranes 118 are well known in the art. Membrane 118 can be an amphiphilic layer or a solid layer. An amphiphilic layer is a layer formed by amphiphilic molecules, such as phospholipids, which have both hydrophilic and lipophilic properties. Amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles forming a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Copolymers can be triblock, tetrablock or pentablock copolymers. Membrane 118 can be a triblock or diblock copolymer membrane.

[0259] Membranes 118 formed from block copolymers have several advantages over biological lipid membranes. Because the triblock copolymers are synthetic, the exact construction can be carefully controlled to provide the correct chain length and properties required to form membranes and interact with pores and other proteins.

[0260] Block copolymers can also be constructed from subunits that are not classified as lipid submaterials, for example, hydrophobic polymers can be prepared from siloxanes or other non-hydrocarbon monomers. The hydrophilic subsegments of the block copolymers can also have low protein binding properties, which allows the production of membranes that are highly resistant when exposed to raw biological samples. 118 Such head group units can also be derived from non-classical lipid head groups.

[0261] Compared to biological lipid membranes, triblock copolymer membranes also have increased mechanical and environmental stability, such as much higher operating temperature or pH ranges.The synthetic nature of block copolymers provides a platform for tailoring polymer-based membranes for a wide range of applications.

[0262] The membrane 118 may be one of the membranes disclosed in WO 2014 / 064443 or WO 2014 / 064444, which are incorporated herein by reference in their entirety. These documents also disclose suitable polymers.

[0263] The amphiphilic molecules can be chemically modified or functionalized to facilitate coupling to polynucleotides.

[0264] The amphiphilic layer can be a single layer or a double layer. 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 posts such that the peripheral area of ​​the amphiphilic layer (where it is connected to the posts) is higher than the area of ​​the amphiphilic layer. This can allow the microparticles to travel, move, slide, or roll along the membrane as described above.

[0265] The membrane 118 may be a lipid bilayer. Suitable lipid bilayers are disclosed in WO2008 / 102121, WO2009 / 077734 and WO2006 / 100484.

[0266] Methods for forming lipid bilayers are known in the art. Lipid bilayers are typically formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA, 1972; 69: 3561-3566), in which a lipid monolayer is carried on an aqueous solution / air interface across an orifice perpendicular to the interface.

[0267] The solid state layer can be formed from both organic and inorganic materials, including but not limited to: microelectronic materials, insulating materials (such as Si3N4, Al2O3 and SiO), organic and inorganic polymers (such as polyamide), plastics (such as ) or elastomers (such as two-component addition-cured silicone rubber) and glass. The solid-state layer can be formed from graphene. Suitable graphene layers are disclosed in WO 2009 / 035647. Yusko et al., Nature Nanotechnology, 2011;6:253-260 and U.S. Patent Application No. 2013 / 0048499 describe protein delivery to transmembrane pores in a solid-state layer without the use of microparticles.

[0268] Any transmembrane pore can be used. Nanopore 116 can be biological or artificial. Suitable nanopores 116 include, but are not limited to, protein pores, polynucleotide pores, and solid-state pores. Nanopore 116 can be a DNA origami pore (Langecker et al., Science, 2012;338:932-936).

[0269] The transmembrane pore can be a transmembrane protein pore. A transmembrane protein pore is a polypeptide or a collection of polypeptides that allows hydrated ions, such as byproducts of polynucleotides treated with a polymerase, to flow from one side of the 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 side. The transmembrane protein pore can allow polynucleotides to flow from one side of a membrane 118 (such as a triblock copolymer membrane) to the other side. The transmembrane protein pore can allow polynucleotides, such as DNA or RNA, to move through the nanopore 116.

[0270] Transmembrane protein pores can be monomeric or oligomeric. 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 hexamer, heptamer, octamer, or nonamer. The pore can be a homo-oligomer or a hetero-oligomer.

[0271] Transmembrane protein pores typically include a barrel or channel through which ions can flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane beta barrel or channel or a transmembrane alpha-helical bundle or channel. The barrel or channel of a transmembrane protein pore typically includes amino acids that promote interaction with nucleotides, polynucleotides, or nucleic acids. These amino acids can be located near the constriction of the barrel or channel. Transmembrane protein pores typically include 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 promote interaction between the pore and the nucleotides, polynucleotides, or nucleic acids.

[0272] The transmembrane protein pore used according to 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), lysin, CsgG, ClyA, Sp1, and the hemolytic protein fragaceatoxin C (FraC). The transmembrane protein pore may be derived from CsgG. Suitable pores derived from CsgG are disclosed in WO2016 / 034591. The transmembrane pore may be derived from lysin. Suitable pores derived from lysin are disclosed in WO2013 / 153359.

[0273] The analyte (including, for example, a protein, peptide, small molecule, polypeptide, polynucleotide) can be present in the analyte. The analyte can be any suitable sample. The analyte can be a biological sample. Any of the embodiments of the methods described herein can be performed in vitro on an analyte obtained or extracted from any organism or microorganism. The organism or microorganism is typically an archaeon, a prokaryote, or a eukaryote, and typically belongs to one of the five kingdoms: Plantae, Animalia, Fungi, Prokaryotes, and Protists. In some embodiments, the various aspects of the methods described herein can be performed in vitro on an analyte obtained or extracted from any virus.

[0274] The analyte can be a fluid sample. The analyte can include a body fluid. The body fluid can be obtained from a human or an animal. The human or animal may have, be suspected of having, or be at risk of having a disease. The analyte can be urine, lymph, saliva, mucus, semen, or amniotic fluid, but can also be whole blood, plasma, or serum. Typically, the analyte is of human origin, but alternatively, it can be from another mammal, such as a commercially raised animal, such as a horse, cattle, sheep, or pig, or alternatively, it can be a pet, such as a cat or dog.

[0275] Alternatively, the analyte may be of plant origin.

[0276] The analyte can be a non-biological sample. The non-biological sample can be a fluid sample. An ionic salt (such as potassium chloride) can be added to the sample to affect the ion flow through the nanopore.

[0277] A polynucleotide may be single-stranded or double-stranded. At least a portion of a polynucleotide may be double-stranded.

[0278] Polynucleotides can be nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Polynucleotides can include an RNA chain hybridized to a DNA chain. Polynucleotides 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. Polynucleotides can be of any length.

[0279] Any number of polynucleotides can be studied. For example, the method can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If two or more polynucleotides are characterized, they can be different polynucleotides or two instances of the same polynucleotide.

[0280] Polynucleotides may be naturally occurring or artificial.

[0281] The method may involve measuring two, three, four, or five or more properties of a polynucleotide. The one or more properties may 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.

[0282] For (iii), the sequence of the polynucleotide can be determined as described above. Suitable sequencing methods, particularly those using electrical measurements, are described in the following literature: Stoddart D et al., Proc Natl Acad Sci, 12; 106(19): 7702-7; Lieberman KR et al., J Am Chem Soc. 2010; 132(50): 17961-72; and International Application WO 2000 / 28312.

[0283] Secondary structure can be measured in a variety of ways. For example, if the method involves electrical measurements, changes in residence time or changes in ionic current flowing through the pore can be used to measure secondary structure. This allows for the differentiation of regions of single-stranded and double-stranded polynucleotides.

[0284] The presence or absence of any modification can be measured. The method may include determining whether the polynucleotide is modified by methylation, oxidation, or damage using one or more proteins or using one or more markers, tags, or spacers. Specific modifications will result in specific interactions with the pore, which can be measured using the methods described below.

[0285] In some embodiments of various aspects described herein, the method may comprise further characterising the target polynucleotide.When the target polynucleotide is in contact with the pore, one or more measurements are taken as the polynucleotide moves relative to the pore, which are indicative of one or more characteristics of the target polynucleotide.

[0286] 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, or damage using one or more proteins or using one or more markers, tags, or spacers.

[0287] Also provided is a kit for characterizing a target polynucleotide. The kit includes components of the pores and membranes disclosed herein. The membrane can be formed from the components. The pores can be present in the membrane. The kit can include components of any of the membranes disclosed above, such as an amphiphilic layer or a triblock copolymer membrane.

[0288] The present invention also provides a device for characterizing a target analyte (e.g., a target polynucleotide). The device comprises a plurality of pores and a plurality of membranes as disclosed herein. The plurality of pores are present in the plurality of membranes. The number of pores and membranes can be equal. Each membrane can have a single pore.

[0289] Devices for characterizing target analytes may include arrays of pores as disclosed herein in a plurality of membranes.

[0290] The device may further include instructions for performing the method. The device may be any conventional device for analyte analysis, such as an array or chip. Any of the embodiments discussed above with reference to the method are also applicable to the device of the present invention. The device may further include any of the features present in the kit as disclosed herein.

[0291] The apparatus may be configured to perform the method as disclosed herein.

[0292] The apparatus may include a sensor device capable of supporting a plurality of wells and a membrane and operable to perform analyte characterization using the wells and the membrane; and at least one port for delivering a material for performing the characterization.

[0293] Alternatively, the apparatus may comprise a sensor device capable of supporting a plurality of wells and a membrane and operable to perform analyte characterisation using the wells and membrane; and at least one reservoir for holding material for performing the characterisation.

[0294] The apparatus may include a sensor device capable of supporting the membrane and the plurality of pores and membranes and operable to perform analyte characterization using the pores and membranes; at least one reservoir for holding material for performing the characterization; a fluid system configured to controllably provide material from the at least one reservoir to the sensor device; and one or more containers for containing corresponding samples, the fluid system being configured to selectively provide analytes from the one or more containers to the sensor device.

[0295] The apparatus may be any of those described in WO2009 / 077734, WO2010 / 122293, WO2011 / 067559 or WO00 / 28312.

[0296] Control of analyte movement relative to the nanopore (e.g., translocation speed, analyte rejection, etc.) can be managed by the systems and methods disclosed in WO 2016 / 059427, which is incorporated herein by reference in its entirety. Rejection of the analyte by the nanopore sensor can include ejection of the analyte from the nanopore.

[0297] According to the teachings herein, the features described above and in the accompanying drawings are interchangeable and compatible. The present invention has been described above by way of example only and can be modified within the spirit and scope of the present invention, which extends to equivalents of the features described and combinations of one or more of the features described herein. The present invention also lies in any individual feature described or suggested herein.

[0298] Feature List:

[0299]

[0300]

Claims

1. A device for performing nanopore sensing, the device comprising: a structure (100) arranged to separate an analyte reservoir from an outlet chamber, the structure (100) comprising an array of nanopore structures (102), each nanopore structure (102) comprising a passageway through the structure (100) for enabling fluid connection between the analyte reservoir and the outlet chamber; a drive electrode connected in the analyte reservoir and the outlet chamber, respectively, to impose a potential difference across the pathway; electrical transduction elements, each element being connected to the passage of a corresponding nanopore structure (102) to measure the fluid potential at the electrical transduction element in the nanopore structure (102); as well as control terminals, each control terminal being connected to a respective nanopore structure (102) for applying a control signal to change the potential difference across the nanopore structure (102), wherein the structure (100) has: a nanoporous layer incorporating an array of pores (142), each pore (142) having a membrane spanning the pore (142) and having a nanopore (116) formed in the membrane, and each pore (142) having a pore outlet; as well as a base layer incorporating channels, wherein the nanoporous layer and the base layer are sandwiched together such that the pores (142) align with corresponding channels to define the passages, wherein each hole (142) is located at a first end of a corresponding passage, wherein each of the electrical transducer elements is configured on a side of the corresponding hole (142) opposite to the corresponding nanopore (116), wherein the pores (142) are fluidically connected to the channel via the pore outlets, the pore outlets being arranged on a side of the respective pores (142) opposite to the respective nanopores (116), The structure (100) further comprises a control electrode, each control electrode is exposed to a passage of a corresponding nanopore structure (102), and each control terminal is connected to the control electrode of the corresponding nanopore structure (102).

2. The device according to claim 1, wherein the electrical transduction element associated with each nanopore structure (102) and the control terminal are directly connected.

3. An apparatus according to claim 1 or 2, wherein the terminals are configured to apply a control signal to change the potential difference across the nanopore structure (102) in response to a measurement of the fluid potential at the electrical transduction element at each respective nanopore structure (102). 4 . The apparatus according to claim 1 , wherein the applying of the control signal is configured to change a potential difference between at least one of the control terminals and at least one of the drive electrodes.

5. The device according to claim 1 or 2, wherein the control signal is connectable to a plurality of the nanopore structures (102) to simultaneously change the potential difference between the connected control terminal and at least one of the drive electrodes. The device according to claim 1 , wherein the electrical transducing element can be isolated from the measuring circuit. The apparatus of claim 6 , wherein the electrical transduction element is capable of being isolated prior to the application of the control signal.

8. The device of claim 1 or 2, wherein the nanopore structure (102) comprises a nanopore (116).

9. The apparatus of claim 8, wherein the control signal is applied to change the potential difference across the nanopore (116) so as to: unblocking the passage of the nanopore (116) when the device detects that the analyte is blocked; rejecting the analyte being measured; and / or The direction and / or speed of analyte translocation through the nanopore (116) is altered.

10. The device according to claim 1 or 2, wherein the array has electronic circuits, each electronic circuit being associated with a respective nanopore structure (102) and connected to the electrical transduction element, each electronic circuit being configured to modify and / or process a signal received from the electrical transduction element.

11. The device according to claim 10, wherein each electronic circuit is associated with a set of nanopore structures (102).

12. A device according to claim 1 or 2, wherein the array has a control circuit, each control circuit being associated with a corresponding nanopore structure (102) and connected to the control terminal and / or the electrical transduction element, the control circuit being configured to change the potential imposed by the drive electrode at the corresponding nanopore structure (102) in response to a signal.

13. The apparatus of claim 12, wherein each control circuit is associated with a group of nanopore structures (102).

14. The device according to claim 13, wherein at least one of the electrical transduction element, the control circuit or the control terminals is disposed on or between an outer surface of the structure (100).

Images