Nanopore sensor device

Abstract

A sensor device (902) for a nanopore sensor, the sensor device (902) comprising: an insulating substrate (903); one or more wells (908) for containing a fluid; wherein the one or more wells (908) are formed on a first side of the substrate (903); a sensor electrode (910) for detecting an ionic current in each of the one or more wells (908); wherein the sensor electrode (910) is formed on the first side of the substrate (903) at the base of the one or more wells (910); one or more through holes (912) extending through the substrate (903); wherein the one or more through holes (912) are connected to the sensor electrode (910); wherein the one or more through holes (912) each comprise a conductive cap (950) at one or both ends of the through hole (912).

Description

Technical Field

[0001] The present invention relates to a nanopore sensor device used in a nanopore sensing apparatus, and in particular to a nanopore sensor device having an insulating substrate. Background Art

[0002] The use of nanopore devices to sense interactions with molecular entities, such as polynucleotides, is a powerful technology that has recently undergone significant development. Nanopore devices comprising arrays of nanopore sensing elements have been developed to enhance data collection by allowing multiple nanopores to sense interactions in parallel, typically from the same sample or analyte.

[0003] Nanopore devices can generally use electrical signals across the nanopore channel to generate measurement signals that are interpreted to sense and / or characterize molecular entities as they interact with the nanopore. Typically, the electrical signal is applied as a potential difference or current across the array of nanopore channels, which provides a meaningful measurement signal to be interpreted. The measurement can include, for example, one of ionic current, resistance, or voltage.

[0004] The array of sensing elements can be mounted on a substrate, typically made of a semiconductor (e.g., crystalline silicon). However, forming vias through the substrate to connect to signal processing circuitry (typically accomplished by doping the semiconductor) is difficult and expensive. Furthermore, the silicon substrate introduces parasitic capacitance, which adds noise to the signal. As will be appreciated, the level of the measured signal can be sensitive to this noise, potentially affecting the accuracy of sensing and / or characterizing the molecular entity being analyzed. Summary of the Invention

[0005] It is an object of the present invention to provide an improved nanopore sensor device.

[0006] When viewed from a first aspect, the present invention provides a sensor device for use in a nanopore sensing apparatus, the sensor device comprising:

[0007] insulating substrate;

[0008] one or more wells for containing a fluid;

[0009] wherein one or more wells are formed on a first side of the substrate;

[0010] one or more sensor electrodes for detecting ion current in the one or more traps;

[0011] wherein the one or more sensor electrodes are formed on the first side of the substrate at the base of the one or more wells;

[0012] one or more through-holes extending through the substrate;

[0013] wherein the one or more through-holes are connected to one or more sensor electrodes;

[0014] The one or more through-holes each include a conductive cap at one or both ends of the through-hole.

[0015] The present invention provides a sensor device for use in a nanopore sensing device. Such a nanopore sensing device may include, for example, a sensor device and a detection (eg, signal processing) circuit.

[0016] The sensor device includes one or more wells formed on one side of an (e.g., electrically) insulating substrate. The wells are arranged to contain a fluid (e.g., an ionic solution) in which the molecular entity to be detected can be provided. The sensor device also includes one or more sensor electrodes formed at the base of the one or more wells on the same (first) side of the substrate as the wells. The wells and sensor electrodes together may form a "sensing element."

[0017] One or more through-holes extend through the substrate, which are connected to the sensor electrodes. This helps to allow measurement values ​​obtained at the sensor electrodes to be communicated from the sensor device to, for example, detection circuitry.

[0018] One or more through-holes each include a conductive cap at one or both ends of the through-hole. Providing the conductive cap at the ends of the through-hole helps provide a good (e.g., flat) surface on which other structures, such as sensor electrodes, can be formed. This facilitates good (electrical) connection between the through-hole and structures formed on the substrate (e.g., sensor electrodes).

[0019] Furthermore, using a substrate formed of an insulating material helps reduce the parasitic capacitance of the sensor device. This helps to increase the measurement sensitivity and, therefore, the accuracy of detecting molecular entities.

[0020] The sensor device may be any suitable and desired sensor device for use in a nanopore sensing apparatus. The sensor device may, for example, have a detailed construction as disclosed in WO 2009 / 077734 or WO 2014 / 064443, which are incorporated herein by reference in their entirety.

[0021] The nanopore sensing device may be any suitable and desired device for sensing molecular entities (eg, polynucleotides).The nanopore sensing device may include a sensor device and a detection circuit connected to the sensor device.

[0022] The detection circuit is preferably arranged to process an electrical signal output from the sensor device (e.g., a sensing element of the sensor device) (e.g., an electrical signal as measured at a sensor electrode). The detection circuit may be arranged to amplify the electrical signal output from the sensor device. The detection circuit may be arranged to (e.g., control the sensing element and) apply a bias signal to the sensing element (e.g., a sensor electrode of the sensing element) to bias the sensor electrode relative to one or more reference electrodes.

[0023] The reference electrode can be a single (e.g., common) reference electrode or a respective reference electrode for each sensor electrode, for example, located in a sensing element (e.g., a well of a sensing element). For example, providing a respective reference electrode for each sensor electrode instead of a single common reference electrode can help reduce the cost of the device.

[0024] A nanopore sensing device (e.g., a detection circuit of the nanopore sensing device) can include a data processor. The data processor can be implemented in any suitable and desired manner, for example, the data processor can include, for example, an application-specific integrated circuit (ASIC) configured to perform nanopore sensing. The design of the detection circuit (e.g., a data processor of the detection circuit) and its functionality can be, for example, as described in WO 2020 / 109800, which is incorporated herein by reference in its entirety.

[0025] In some embodiments, the detection circuit (e.g., a data processor of the detection circuit) can be arranged to control a potential applied to a sensor electrode (e.g., each of the sensor electrodes). In some embodiments, the detection circuit (e.g., a data processor of the detection circuit) can be arranged to measure, digitize, and / or output a current (the current flowing into the well and converted by the sensor electrode).

[0026] The sensor device can be connected to the detection circuit (e.g., a data processor of the detection circuit) in any suitable and desired manner. In some embodiments, the nanopore sensing apparatus includes an interposer, wherein the interposer is connected to the sensor device and the detection circuit, wherein the interposer is arranged to communicate signals from the sensor device to the detection circuit. The interposer may, for example, comprise a printed circuit board (PCB) or similar component.

[0027] The detection circuit may be arranged to output the measured (eg amplified) signal to an analysis system.

[0028] The insulating substrate of the sensor device can be formed from any suitable and desired insulating (e.g., electrically insulating) (non-conductive) material. The insulating substrate can include a dielectric substrate. The insulating substrate can be formed from a ceramic material (e.g., aluminum oxide, silicon nitride, quartz), an amorphous solid, a crystal, a non-crystalline material, and / or a mineral (e.g., sapphire or sapphire glass).

[0029] In some embodiments, the insulating substrate includes (e.g., consists of) a glass substrate. Providing a glass substrate helps reduce parasitic capacitance of the sensor device due to its high resistivity. Glass also facilitates easier and less expensive formation of through-substrate vias due to its mechanical properties, for example, compared to doped vias formed in semiconductor substrates. This is because vias in glass substrates can be formed mechanically (e.g., using a laser) and / or chemically (e.g., by etching).

[0030] Any suitable and desired glass type can be used for the glass substrate.In some embodiments, the glass of the substrate comprises borosilicate glass.

[0031] One or more wells are formed on the first side of the substrate for receiving a fluid. The wells may be formed and arranged in any suitable and desired manner. The sensor device may include one or more (e.g., a plurality of) walls formed on the first side of the substrate, wherein the walls define the one or more wells (i.e., define the one or more wells between the one or more walls).

[0032] In some embodiments, the sensor device includes a support structure formed on a first side of a substrate, wherein the support structure defines one or more wells (eg, one or more wells of one or more walls).

[0033] The walls and / or support structures may be formed from any suitable and desired material. The walls and / or support structures may be formed from an insulating material or a stack of materials (e.g., a laminated stack). In some embodiments, the walls and / or support structures are formed as photoresist structures.

[0034] The walls and / or support structure may be arranged to support a membrane above the wells (eg, each of the wells). The membranes preferably (eg, each) contain a nanopore inserted in the membrane. The membranes may include amphiphilic molecules, such as lipids or polymers.

[0035] The support structure and the membrane supported on the well may, for example, take the form as described in WO 2014 / 064443 and WO 2021 / 255414, which are incorporated herein by reference in their entirety.

[0036] One or more sensor electrodes are formed on the first side of the substrate at the base of one or more wells (e.g., each of the wells). Thus, in some embodiments, (e.g., each) well contains (only) a single sensor electrode. The sensor electrodes are arranged (when the sensor device is in use) to detect ionic current in (e.g., the corresponding) well, e.g., to sense and / or characterize a molecular entity when the molecular entity interacts with a nanopore supported by the well.

[0037] The sensor electrodes can be arranged at the base of one or more wells in any suitable and desired manner. In some embodiments, the sensor electrodes extend over the entire base of the (e.g., corresponding) well. The sensor electrodes can extend over an area of ​​the substrate corresponding to the base of the (e.g., corresponding) well, or they can extend over an area of ​​the substrate that is larger than (the area of) the base of the (e.g., corresponding) well, such as extending below the walls (e.g., the material forming the walls) of one or more wells.

[0038] In some embodiments, the sensor electrodes extend partially over the base of the (eg, corresponding) well.Thus, in some embodiments, the first side of the substrate is exposed at the base of one or more wells.

[0039] In some embodiments, one or more sensor electrodes are formed on (eg, connected to) a corresponding conductive cover. Thus, in these embodiments, each of the one or more through-holes includes a conductive cover proximate the first side of the substrate.

[0040] The sensor electrodes can have any suitable and desired structure. In some embodiments, one or more sensor electrodes (e.g., each of the one or more sensor electrodes) include an electrode base layer proximate to the substrate and an electrode coating exposed to the well. Preferably, the electrode base layer is formed on (e.g., connected to) the (corresponding) conductive cover. In some embodiments, the electrode base layer at least partially (e.g., completely) covers the conductive cover.

[0041] The electrode base layer may be provided, for example, as a "seed" layer, eg, a material that adheres well to the substrate and / or does not interfere with the electrochemical potential of the interface between the solution in the well and the electrode coating.

[0042] The electrode coating may be formed of a material that is suitable for measuring ion current (eg, sensitive to measuring ion current) and that, for example, can adhere well to the electrode base layer.

[0043] In some embodiments, the electrode coating at least partially (eg, completely) covers the electrode base layer. In some embodiments, the electrode coating extends over the entire base of the (eg, corresponding) well, eg, even though the electrode coating may not necessarily extend over the entire electrode base.

[0044] The sensor electrodes (eg, the electrode base layer and the electrode coating) can be formed from any suitable and desired material. In some embodiments, the electrode base layer comprises a transition metal, such as titanium.

[0045] In some embodiments, the electrode coating has a greater electrical conductivity than the electrode base layer. This can help the electrode coating be more sensitive to measuring ion current in the well.

[0046] In some embodiments, the electrode coating is formed from a less reactive material than the electrode base layer. Forming the electrode coating (which is exposed to the solution in the well (during use)) from a less reactive (e.g., inert) material than the electrode base layer helps reduce any (e.g., electrochemical) interaction between the electrode coating and the solution in the well and helps prevent corrosion of the electrode coating.

[0047] In some embodiments, the electrode coating comprises a noble metal (eg, gold or platinum) and / or a transition metal (eg, palladium).

[0048] In some embodiments, the sensor electrode comprises a silver-silver chloride electrode, such as a silver chloride electrode, for example, a silver substrate layer coated with silver chloride. This type of electrode may be suitable when the ion solution used in the trap comprises a chloride solution. The silver chloride coating can be formed in situ on the silver electrode in the chloride solution.

[0049] The sensor device has one or more through-holes extending through the substrate, and the through-holes are connected to one or more sensor electrodes. The one or more through-holes (e.g., each through-hole) can extend from a first side of the substrate to a second side of the substrate, e.g., the through-holes extend substantially perpendicular to the plane of the substrate.

[0050] The through holes may be arranged in any suitable and desired manner.In some embodiments, the through holes are defined by walls passing through the substrate.

[0051] In some embodiments, one or more vias (eg, each via) comprises a conductor forming a conductive path through the via. Preferably, the conductor is connected to a (corresponding) sensor electrode, eg, to form a conductive path from the sensor electrode through the via.

[0052] The conductors forming the conductive paths through the through-holes can be arranged in the through-holes in any suitable and desired manner to form the conductive paths. In some embodiments, the conductors extend through the through-holes. In some embodiments, the conductors comprise a conductive material, e.g., one or more through-holes are each (at least partially) filled with a conductive material.

[0053] In some embodiments, the conductor comprises a wire, filament, or ribbon, for example, disposed substantially in the center of the through-hole (e.g., away from the walls of the through-hole). In some embodiments, the conductor comprises a conductive cylinder that at least partially lines the through-hole (e.g., the walls of the through-hole).

[0054] The conductor can comprise any suitable and desired (conductive) material. In some embodiments, the conductor (e.g., conductive material) comprises (e.g., consists of) a transition metal, such as copper. In some embodiments, the conductive cap is formed from the same material (e.g., conductive material) as the conductor (material).

[0055] In some embodiments, a material is disposed (e.g., in each via) to retain the conductor within the via. The material disposed to retain the conductor within the via can be any suitable and desired material. In some embodiments, the material disposed to retain the conductor within the via is different from the material of the conductor. In some embodiments, the material disposed to retain the conductor within the via is different (and / or, for example, substantially different) from the insulating material of the substrate.

[0056] In some embodiments, the material arranged to retain the conductor in the through-hole substantially fills the through-hole. In some embodiments, the material arranged to retain the conductor in the through-hole comprises a sealant, such as an adhesive. Preferably, the material arranged to retain the conductor in the through-hole is arranged to substantially seal the through-hole (e.g., between a first side of the through-hole and a second side of the through-hole).

[0057] In some embodiments, when the conductor comprises a conductive cylinder at least partially lining the through-hole (e.g., a wall of the through-hole), the material arranged to retain the conductor in the through-hole substantially fills the conductive cylinder. Preferably, the material arranged to retain the conductor in the through-hole is arranged to substantially seal the conductive cylinder in the through-hole.

[0058] In some embodiments, the sensor device includes one or more contacts formed on the second side of the substrate. Preferably, the one or more contacts are connected to one or more sensor contacts (e.g., each sensor electrode is connected to a corresponding contact) through one or more through-holes (e.g., corresponding through-holes) in the through-holes.

[0059] Thus, one or more (electrical) contacts may be formed on the second (opposite) side of the substrate.Preferably, the conductors are connected to the (respective) contacts, eg to form an electrically conductive path (eg from a sensor electrode) through the via to the contact.

[0060] In some embodiments, one or more contacts are formed on (eg, connected to) a corresponding conductive cover. Thus, in these embodiments, each of the one or more through-holes includes a conductive cover proximate the second side of the substrate.

[0061] The contacts can have any suitable and desired structure. In some embodiments, one or more contacts (e.g., each of the one or more contacts) include a contact base layer proximate to the substrate and a contact overcoat. Preferably, the contact base layer is formed on (e.g., connected to) the (corresponding) conductive cover. In some embodiments, the contact base layer at least partially (e.g., completely) covers the conductive cover.

[0062] The contact base layer can be provided, for example, as a "seed" layer, which is a material that adheres well to a substrate. The contact overcoat can be formed of a material suitable for forming an electrical connection, such as with a sensor device or another component in a nanopore sensing apparatus.

[0063] In some embodiments, the contact overcoat at least partially (eg, completely) covers the electrode base layer.

[0064] The contacts are arranged to be suitable for connection to other components (e.g., components) of the nanopore sensing device. In some embodiments, the contacts are arranged to make a permanent connection, for example, using solder. In some embodiments, the contacts can be formed into a shape (e.g., including a recess) for receiving a solder ball.

[0065] In some embodiments, the contacts are arranged to make a temporary (detachable) connection, for example using spring contacts. In some embodiments, the contacts may be shaped to receive the spring contacts (eg, including a protrusion).

[0066] The contacts (eg, contact base layer and contact overcoat) can be formed of any suitable and desired material. In some embodiments, the contact base layer comprises a transition metal, such as copper and / or titanium.

[0067] In some embodiments, the contact overcoat comprises a noble metal, such as gold.

[0068] In some embodiments, the contact overcoat layer has a greater electrical conductivity than the contact base layer. This can help the contact overcoat layer form a good electrical connection, for example, with a sensor device or another component in a nanopore sensing apparatus.

[0069] In some embodiments, one or more contacts include a contact intermediate layer between the contact base layer and the contact outer coating. This can help form the shape of the contact and form an effective connection between the contact base layer and the contact outer coating.

[0070] The contact intermediate layer can comprise any suitable and desired material. In some embodiments, the contact intermediate layer comprises a transition metal, such as nickel or palladium. Nickel can be used when the contact is arranged to make a permanent connection. Palladium can be used when the contact is arranged to make a temporary connection.

[0071] The contact middle layer and the contact outer layer can be in the form of an electroless nickel immersion gold (ENIG) or electroless palladium immersion gold (EPIG) coating.

[0072] In some embodiments, the contact outer coating has a greater electrical conductivity than the contact intermediate layer. Again, this can help the contact outer coating form a good electrical connection, such as with a sensor device or another component in a nanopore sensing apparatus.

[0073] In some embodiments, the sensor device includes an insulating layer on the second side of the substrate. The insulating layer may at least partially (e.g., substantially completely) surround one or more contacts. The insulating layer may contact the second side of the substrate. If the sensor device includes multiple contacts, the insulating layer may be disposed between the contacts.

[0074] The insulating layer may comprise any suitable and desired insulating material, for example silicon dioxide, a fluoropolymer, or a thermoplastic polymer such as polybenzoxazole (PBO).

[0075] The one or more through-holes each include a conductive cover at one or both ends of the through-hole. Preferably, the through-hole (e.g., the conductor of the through-hole) is connected to the conductive cover at one or both ends of the through-hole. Preferably, the conductive cover is connected to a (corresponding) sensor electrode and / or a (corresponding) contact.

[0076] Thus, one or more through-holes (e.g., conductors) can (e.g., each) be connected (via the conductive cover) to a (corresponding) sensor electrode and / or a (corresponding) contact (e.g., connect (e.g., each) sensor electrode to a (e.g., corresponding) contact). The conductive cover can be connected to a (corresponding) electrode base layer and / or a (corresponding) contact base layer.

[0077] The conductive cap may comprise any suitable and desired (conductive) material. In some embodiments, the conductive cap comprises (e.g., consists of) a transition metal, such as copper. In some embodiments, the conductive cap is formed of the same material as the (conductive) material of the conductor.

[0078] The conductive cap can have any suitable and desired geometry (i.e., size and shape). In some embodiments, the diameter of the conductive cap is substantially equal to the diameter of the (corresponding) through-hole. Preferably, the conductive cap is coaxial with the (corresponding) through-hole. In some embodiments, the (exterior) surface of the conductive cap is substantially (co)planar with the surface of the substrate.

[0079] It should be understood that in some embodiments, the sensor device includes multiple sensor elements, i.e., multiple wells, multiple sensor electrodes, and multiple through-holes. The multiple sensor elements may all be arranged on the same (e.g., integral) substrate. Thus, in some embodiments, the sensor device includes:

[0080] a plurality of wells for containing fluids;

[0081] wherein a plurality of wells are formed on a first side of the substrate;

[0082] a plurality of sensor electrodes for detecting ion currents in the plurality of traps;

[0083] wherein a plurality of sensor electrodes are formed on the first side of the substrate at the base of the plurality of wells;

[0084] a plurality of through-holes extending through the substrate;

[0085] wherein the plurality of through-holes are connected to the plurality of sensor electrodes;

[0086] Each of the plurality of through holes includes a conductive cover at one or both ends of the through hole. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Certain embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0088] Figure 1 shows a schematic diagram of a nanopore array device;

[0089] Figure 2 shows a schematic cross-sectional view of a portion of a nanopore array device;

[0090] Figure 3 Schematic cross-sectional views showing various components of a nanopore sensor according to an embodiment of the present invention;

[0091] Figure 4 shows a schematic cross-sectional view of a sensor device according to an embodiment of the present invention;

[0092] Figure 5 shows a schematic cross-sectional view of electrodes on the front side of a substrate according to an embodiment of the present invention;

[0093] Figure 6 shows a schematic cross-sectional view of a sensor device according to an embodiment of the present invention;

[0094] Figure 7 Shown Figure 6 A plan view of the electrode shown;

[0095] Figure 8 shows a schematic cross-sectional view of electrodes on the front side of a substrate according to an embodiment of the present invention;

[0096] Figure 9 shows a schematic cross-sectional view of a sensor device according to another embodiment of the present invention; and

[0097] Figure 10 A schematic cross-sectional view of substrate contacts on the back side of a substrate according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0098] Various embodiments of the present invention will now be described in the context of nanopore sensors, which can be used to sense molecular entities.

[0099] Figure 1A schematic diagram of a nanopore array device 1 for sensing interactions of molecular entities is shown. The nanopore array device 1 comprises a sensing arrangement 2 comprising a sensor device 3 and a detection circuit 4 connected to the sensor device 3 .

[0100] The sensor device 3 comprises an array of sensing elements 30, each supporting a respective nanopore channel capable of interacting with a molecular entity. The sensing elements 30 comprise respective sensor electrodes 31. In use, each sensing element 30 outputs an electrical measurement at its sensor electrode 31 that is dependent on the interaction of the molecular entity with the nanopore. Figure 1 It is shown schematically in FIG, but can have a variety of configurations, some non-limiting examples of which are as follows.

[0101] In one example, the sensor device 3 may have Figure 2 The shape shown, Figure 2 Schematic cross-sectional view of the sensor device 3 is shown. Figure 2 As shown, the sensor device 3 includes an array of sensing elements 30. Each sensing element 30 includes a membrane 32 supported on a well 33 formed in a support structure 34. A nanopore 35 is inserted into the membrane 32 through each well 33, thereby providing a channel extending from one side of the membrane to the other. The membrane 32 may contain amphiphilic molecules, such as lipids or polymers.

[0102] Each membrane 32 seals a corresponding well 33 from a liquid sample ("cis") chamber 36, which extends through the array of sensing elements 30 and is in fluidic communication with each nanopore 35. Each well 33 has a sensor electrode 31 at its ("trans") base. A common electrode 37 is disposed in the sample chamber 36 to provide a common reference signal (typically a potential or voltage) to each sensing element 30. In some embodiments, a corresponding reference electrode can be provided for each sensor electrode, for example, in the well of the sensing element. A liquid sample can be provided in each well.

[0103] In use, the sample chamber 36 receives a sample containing an ionic solution and molecular entities that interact with the nanopores 35 of the sensing element 30. In the sensing mode of the sensing device 2, an ionic current flows from the common electrode 37 through the corresponding nanopores 35 to the sensor electrodes 31. Molecules within the nanopores 35 (e.g., molecules that pass through the nanopores) restrict the flow of ions through the nanopores and, therefore, modulate the ionic current measured over time by the sensor electrodes 31. This can be used to characterize (e.g., identify) the molecules.

[0104] For clarity, Figure 2Two sensing elements 30 are shown in FIG, but generally any number of sensing elements 30 may be provided. Generally, a large number of sensing elements 30 may be provided to optimize the data collection rate, such as 256, 1024, 4096 or more sensing elements 30.

[0105] The sensor device 3 may, for example, have a detailed construction as disclosed in WO 2009 / 077734 or WO 2014 / 064443, which are incorporated herein by reference in their entirety.

[0106] The associated elements of the nanopore channel 35 and the sensing element 30 may be as follows, but are not limited to: Figure 2 Example shown.

[0107] Nanopore channel 35 is a pore, typically having a size on the order of nanometers. In embodiments where the molecular entity is a polymer that interacts with the nanopore channel 35 while being transported therethrough, the nanopore channel 35 is of a suitable size to allow the polymer to pass therethrough.

[0108] The use of nanopores to characterize molecular entities is well known in the art. Exemplary nanopores used in the present invention include protein pores, origami pores, and solid-state pores. Protein pores can be wild-type or modified. Transmembrane pores can be derived from or based on, for example, Msp, α-hemolysin (α-HL), hemolysin, CsgG, ClyA, Sp1, and the hemolytic protein fragaceatoxin C (FraC). Examples of materials that can provide solid-state pores are graphene and silicon nitride. The pore size can be such that only one polymer can displace the pore at a time.

[0109] Now we will discuss Figure 1 The arrangement of the detection circuit 4 is shown. The detection circuit 4 is connected to the electrode 31 of each sensor element 30 and has the main function of processing the electrical signal output therefrom. The detection circuit 4 also has the function of controlling the application of a bias signal to each sensor element 30.

[0110] Detection circuit 4 includes multiple detection channels 40. Each detection channel 40 receives an electrical signal from a single sensor electrode 31 and is configured to amplify the electrical signal. Therefore, detection channels 40 are designed to amplify very small currents with sufficient resolution to detect characteristic changes caused by the interaction of interest. Detection channels 40 are also designed with sufficiently high bandwidth to provide the temporal resolution required to detect each such interaction. These limitations necessitate sensitive and therefore expensive components.

[0111] Each detection channel 40 can be similar to the standard single-channel recording apparatus described in Stoddart D et al., Proc Natl Acad Sci USA (May 12, 2009; 106(19):7702-7), Lieberman KR et al., J Am Chem Soc. (Dec 22, 2010; 132(50):17961-72), and WO 2000 / 28312, which are incorporated herein by reference in their entireties. Alternatively, each detection channel 40 can be arranged as described in detail in WO 2010 / 122293, WO 2011 / 067559, or WO 2016 / 181118, which are incorporated herein by reference in their entireties.

[0112] The analyte of interest to be detected by the nanopore can be a polynucleotide, such as DNA or RNA. The analyte can be a polypeptide or a polysaccharide. The number of sensing elements 30 in the array is greater than the number of detection channels 40, and the nanopore array device is operable to obtain polymer measurements from selected sensing elements 30 in a multiplexed manner, particularly an electrically multiplexed manner. This is achieved by providing a switch arrangement 42 between the sensor electrodes 31 of the sensing elements 30 and the detection channels 40.

[0113] For clarity, Figure 1 A simplified example with four sensing elements 30 and two detection channels 40 is shown, but the number of sensor elements 30 and detection channels 40 is typically much larger. For example, for some applications, the sensor device 3 may include a total of 4096 sensing elements 30 and 1024 detection channels 40.

[0114] The switch arrangement 42 may be arranged as described in detail in WO 2010 / 122293. For example, the switch arrangement 42 may include a plurality of 1 to N multiplexers, each multiplexer connected from a detection channel 40 to a set of N sensing elements 30, and may include appropriate hardware (such as latches) to select the state of the switches.

[0115] The nanopore array device 1 can be operated to amplify electrical signals from the sensing elements 30 selected in an electrically multiplexed manner by switching the switch arrangement 42. The detection circuit 4 includes a data processor 5 that receives output signals from the detection channels 40. The data processor 5 acts as a controller that controls the switch arrangement 42 to connect the detection channels 40 to the corresponding sensing elements 30, as further described below.

[0116] In addition, the detection circuit 4 includes a bias control circuit 41 to perform the function of controlling the application of a bias signal to each sensor element 30. The bias control circuit 41 is connected to the common electrode 37 and the sensor electrode 31 of each sensor element 30. The bias signal is selected to bias the sensor electrode 31 relative to the common electrode 37, thereby controlling the translocation of the molecular entity relative to the nanopore 35. Generally, the bias signal provided to a given sensor element 30 can be a driving bias signal that causes translocation to occur at the sensor element 30, or an inhibiting bias signal that inhibits translocation to occur at the sensor element 30.

[0117] The bias control circuit 41 is controlled by the data processor 5. The data processor has an operating mode for the bias control circuit 41. That is, three independent test bias signals are provided to all sensor elements 30, thereby causing an ion current to flow from the common electrode 37 through the nanopore 35 to the sensor electrode 31 of each sensor element 30. The current corresponding to each test signal is recorded in the data processor 5 as an amplified electrical signal.

[0118] The data processor 5 is arranged as follows. It is connected to the output of the detection channel 40 and is supplied with the amplified electrical signal therefrom. The data processor 5 stores and analyzes the amplified electrical signal from the test bias signal to generate a calibrated signal. The data processor 5 also controls other elements of the detection circuitry, including the bias voltage circuit 41 described above and the switch arrangement 42 described below. The data processor 5 forms part of the detection circuitry 4 and, in some examples, may be provided in a common package with the detection circuitry, possibly on a common circuit board. The data processor 5 may be implemented in any suitable form, such as as a processor running an appropriate computer program or as an ASIC (Application Specific Integrated Circuit).

[0119] The data processor 5 of the nanopore array device 1 is connected to an analysis system 6. The data processor 5 also provides the amplified output signal to the analysis system 6. The analysis system 6 performs further analysis on the amplified electrical signal, which is the raw signal representing the measurement result of the property measured at the nanopore. Such an analysis system 6 can, for example, estimate the overall identity of a molecular entity or, in the case of a polymer, the identity of its polymer units. Thus, the analysis system can be configured as a computer device running an appropriate program. Such a computer device can be connected to the data processor 5 of the nanopore array device 1 directly or via a network, for example, within a cloud-based system.

[0120] Figure 3Schematically illustrates a cross section of various components of a sensing device 901 according to an embodiment of the present invention. The sensing device 901 comprises a nanopore sensor device 902 comprising a substrate 903 on which a support structure 904 is formed. For example, the sensor device 902 may be used as Figure 1 and Figure 2 The sensor device 3 in the nanopore array device 1 is shown.

[0121] The support structure 904 includes a plurality of walls 906 defining a plurality of wells 908 therebetween. Figure 3 Only a cross-section is shown in FIG, but it should be understood that the sensor device 902 may include a two-dimensional array of wells 908 distributed over the substrate 903.

[0122] A support structure 904 is formed on the "front" side of substrate 903. Support structure 904 is configured to support a membrane over each of wells 908, wherein the membrane is designed to contain a nanopore inserted therein. Support structure 904 and the membrane supported over each of wells 908 can, for example, take the form described in WO 2014 / 064443 and WO 2021 / 255414, which are incorporated herein by reference in their entireties.

[0123] The well may be filled with an ionic solution (e.g., an aqueous solution). The ionic solution may include a soluble electrode medium, such as ferrocyanide or ferrocyanide. The ionic solution may be as described in WO 2018 / 060740, which is hereby incorporated by reference in its entirety.

[0124] A plurality of sensor electrodes 910 are also formed on the front side of the substrate 903 such that a sensor electrode 910 is disposed at the bottom of each well 908. Each sensor electrode 910 can be used to facilitate measurement of ion current between a common electrode in a corresponding well 908 and the sensor electrode 910.

[0125] The sensor electrode 910 can be made of a material suitable for forming an electrochemical interface and depends on the chemical composition used. Examples include platinum (e.g., used with a soluble redox couple such as ferrous / ferrocyanide mediator) and silver-silver chloride (e.g., used with a chloride ion solution), as described, for example, by Ayub M et al., Electrochimica Acta 55 (2010) 8237-8243, which is incorporated herein by reference in its entirety.

[0126] A plurality of vias 912 are formed through substrate 903 to connect a corresponding plurality of sensor electrodes 910 to a corresponding plurality of substrate contacts 914 on an opposite "back" side of substrate 903. Vias 912 each have a diameter selected, for example, based on the pitch between wells.

[0127] In an exemplary geometry, the distance between the wells ("pitch") (and therefore the distance between the plurality of sensor electrodes 910 and the plurality of substrate contacts 914) can be in the range of 100 μm to 300 μm, for example, approximately 200 μm. The diameter of the through-holes 912 can be in the range of 40 μm to 100 μm, for example, approximately 70 μm. The diameter of the (e.g., circular) sensor electrodes 910 can be in the range of 45 μm to 105 μm, for example, approximately 90 μm.

[0128] The sensing device 901 typically also includes an electrical interposer. This interposer is typically a printed circuit board (PCB) 916, but can also employ "PCB-like" technologies such as ceramic interposers, high-density PCBs (HD-PCBs), or so-called "substrate PCBs." The PCB 916 includes a plurality of input contacts 918 that are connected to a plurality of substrate contacts 914 formed on the substrate 903. This connection can be permanent, such as by permanently bonded electrical contacts, such as solder balls 920 (e.g., Figure 3 As shown), heat-soldered metal (e.g., Cu) posts, or anisotropic conductive film (ACF) tape. Alternatively, the connection can be designed to be disconnected and reconnected multiple times, such as using mechanical spring contacts or similar devices.

[0129] The PCB 916 also includes a plurality of output contacts 922. The plurality of output contacts 922 are connected to the plurality of input contacts 918 via a plurality of tracks 924 formed on the PCB 916.

[0130] The sensing device 901 also includes an application-specific integrated circuit (ASIC) 926, which is designed for nanopore sensing. More details on typical ASIC design and functionality can be found in WO 2020 / 109800, which is incorporated herein by reference in its entirety. In summary, the ASIC performs the following functions:

[0131] 1. Control the potential applied to each of the electrodes 910 in the array; and

[0132] 2. Measure, digitize, and output the current flowing into the plurality of wells 908 and converted by the plurality of electrodes 910.

[0133] ASIC 926 includes a plurality of ASIC contacts 928 for respectively connecting to a plurality of output contacts 922 on PCB 916. It should be understood that in some embodiments, the PCB may be omitted and ASIC 926 may be connected directly to sensor device 902.

[0134] Thus, the plurality of sensor electrodes 910 in the plurality of wells 908 are connected to the ASIC 926 via a plurality of through-holes 912 passing through the substrate 903, a plurality of substrate contacts 914 on the opposite side of the substrate 903, a plurality of input contacts 918 on the PCB 916, a plurality of tracks 924 on the PCB 916, a plurality of output contacts 922 on the PCB 916, and a plurality of ASIC contacts 928. The PCB 916 is configured to match and connect the layout of the plurality of substrate contacts 914 (corresponding to the layout of the plurality of sensor electrodes 910 and the plurality of wells 908) with the plurality of ASIC contacts 28.

[0135] PCB 916 may also include contact pads for other circuit components, connections between circuit components and outputs of ASIC 926, and outputs for connecting to the rest of the nanopore array device. PCB 916 may also house other electronic components, thereby forming the "disposable" portion of sensor device 902. These components may include decoupling capacitors, test points for testing characteristics, non-volatile memory for storing identification and / or calibration parameters, components for regulating the temperature of sensor device 902 (e.g., resistors for Joule heating and / or temperature sensors), analog circuits for supporting the operation of ASIC 926 (e.g., precision voltage references), and the like. In some embodiments, one or more of these components may be provided by (e.g., integrated into) ASIC 926.

[0136] PCB 916 can also be arranged to provide a potential to a common (reference) electrode of the sensor device. This can be via a (e.g., platinum) connecting wire to an external reference electrode in contact with the liquid in the "cis" volume, or via a common electrode provided on sensor device 902 and connected via a through-hole. In embodiments where separate reference electrodes are provided (e.g., for each well), PCB 916 can provide a potential to these reference electrodes in a similar manner, e.g., via corresponding wires or through-holes.

[0137] Although only a single nanopore sensor device 902 (with a single substrate 903) is shown, it should be understood that any number of nanopore sensor devices 902 (and their associated substrates 903) may be provided in the sensing apparatus 901, for example, each device having multiple sensing elements. Multiple nanopore sensor devices 902 may be connected to the same ASIC 926, for example, via the same PCB 916.

[0138] Figure 4 A cross section of a sensor device 930 according to an embodiment of the present invention is schematically shown. The sensor device 930 may be used, for example, Figure 3 Nanopore sensing device 901 is shown.

[0139] and Figure 3 The sensor device shown is similar to Figure 4 The illustrated sensor device 930 includes a substrate 933 having formed thereon a support structure 934 having a plurality of walls 936 defining therebetween a plurality of wells 938. The support structure 934 is formed on the front side of the substrate 933 and is configured to support a membrane designed to contain a nanopore inserted therein, above each of the wells 938.

[0140] A sensor electrode 940 is formed on the front side of the substrate 933 at the bottom of each well 938. A plurality of vias 942 are formed through the substrate 933 to connect a corresponding plurality of sensor electrodes 940 to a corresponding plurality of substrate contacts 944 on the back side of the substrate 933.

[0141] exist Figure 4 In the embodiment shown, substrate 933 is formed of glass. Glass has several physical properties that make it a suitable material for this application, such as:

[0142] a. High resistivity;

[0143] b. Its mechanical properties: good rigidity, flatness and fracture resistance;

[0144] c. Good thermal conductivity, supporting good heat transfer between the ASIC and the wells, which can be important for controlling the temperature of the liquid in the wells;

[0145] d. The thermal expansion coefficient is well matched to that of the PCB and the (usually) photoresist material used to form the well structure;

[0146] e. Compatible with the manufacturing process used to make through holes in the structure;

[0147] f. can be mass-produced; and

[0148] g. Low potential for introducing chemical or biochemical contaminants into the liquid trap.

[0149] Examples of suitable glass materials are borosilicates, such as Asahi Glass AN100, AN Wizus, Corning 1737, Corning 7740, Schott Borofloat 33.

[0150] In some embodiments, the substrate may be formed of an insulating material, including, for example, a ceramic material (eg, alumina, silicon nitride, quartz), an amorphous solid, or a non-crystalline material.

[0151] The support structure 934 is formed on the substrate 933 as a photoresist structure to form a plurality of wells 938. The support structure 934 may be formed of an insulating material (eg, a photoresist structure) or a material stack (eg, a laminate).

[0152] The support structure 934 may, for example, take the form as described in WO 2014 / 064443 and WO 2021 / 255414, which are incorporated herein by reference in their entirety.

[0153] Each through-hole 942 extending through substrate 933 includes a conductive (e.g., copper) cylindrical coating 946 filled with adhesive 948 to seal through-hole 942. Each through-hole 942 has a diameter of approximately 70 μm. The pitch between through-holes 942 (and therefore the spacing between the plurality of sensor electrodes 940 and the plurality of substrate contacts 944) is approximately 200 μm. Optionally, a copper cap 950 is provided at each end of through-hole 942 (proximate each of the front and back sides of substrate 933). This cap forms a flat surface on which sensor electrodes 940 and substrate contacts 944 can be formed.

[0154] The sensor electrode 940 on the front side of the substrate 933 is composed of two layers: a base layer 952 made of titanium and a coating 954 made of platinum. The titanium base layer 952 is tightly adhered to the substrate 933 and thus isolates the non-inert copper cover 950 from the liquid in the corresponding well 938. The platinum coating 954 provides an inert layer with good conductivity for measuring the ion current in the well 938.

[0155] Figure 4 The sensor electrodes 940 shown are substantially circular and have a diameter of approximately 90 μm. The sensor electrodes 940 are substantially symmetrically arranged with respect to the corresponding wells 938 and the corresponding through-holes 942, i.e., each set of wells 938, sensor electrodes 940 and through-holes 942 are substantially coaxial with each other. Figure 3 (and other embodiments) are shown in contrast to the arrangement shown in Figure 3 In the arrangement shown, the sensor electrodes 910 are offset (and non-coaxial) from the corresponding through-holes 912 , and the wells 908 are offset from the center (of the titanium base layer) of the corresponding sensor electrodes 910 .

[0156] In addition, if Figure 4 As shown, the platinum coating 954 completely covers the titanium base layer 952, such that the platinum coating 954 extends around the sides of the titanium base layer 952. The base layer 952 helps adhere the sensor electrode 940 to the substrate 933.

[0157] On the contrary, Figure 3 As shown, the diameter of the platinum coating of the sensor electrode 910 is smaller than that of the corresponding titanium base layer, and the platinum coating is offset from the center of the titanium base layer.

[0158] Therefore, in Figure 3 In the illustrated embodiment, the platinum coating does not completely cover the titanium base layer, although it does extend completely over the base of well 908. This means that the liquid in the well is only exposed to the platinum coating of the sensor electrode, not to the titanium base layer. It also provides (lateral) isolation between the copper in the through-hole 912 and the liquid in the corresponding well 908, which electrochemically facilitates the well 908 and the measurement of ion current.

[0159] In some embodiments, sensor electrode 940 may not extend completely above the base of well 908. As a result, a portion of the liquid in the well may be exposed to substrate 930.

[0160] Substrate contacts 944 on the back side of substrate 933 are each composed of four layers: a base layer 956 made of copper, a first (proximal) intermediate layer 958 made of copper, a second (distal) intermediate layer 960 made of nickel, and an overcoat layer 962 made of gold. The second (distal) intermediate layer 960 and overcoat layer 962 can be applied to the first (proximal) intermediate layer 958 using an electroless nickel immersion gold (EPIG) coating. The copper base layer 956 is connected to substrate 933 and extends over and around the copper cap 950 on through-hole 942. The copper base layer 956 provides a platform for the other layers of substrate contact 944. It should be understood that this structure is merely an example, and other suitable structures for substrate contact 944 are possible.

[0161] An insulating layer 964 (e.g., made of polybenzoxazole (PBO)) may be disposed on the copper base layer 956. Openings are provided in the insulating layer 964 to allow each copper interlayer 958 to contact the corresponding copper base layer 956. The copper interlayer 958 extends over a portion of the insulating layer 964, for example, forming a recess in the center of the copper interlayer 958. A nickel interlayer 960 extends over the entire copper interlayer 958. A gold overcoat 962 covers the entire nickel interlayer 960.

[0162] It should be understood that the structure of substrate contacts 944 may vary, for example, depending on the configuration of the connection (e.g., connection to a PCB or ASIC) and whether the connection is permanent (e.g., using solder) or temporary (e.g., through spring contacts).

[0163] The substrate contacts 944 on the back side of the substrate 933 are configured to connect to input contacts of the PCB (e.g., as shown) through corresponding solder balls. Figure 3938). It will be seen that substrate contacts 944 on the back side of substrate 933 differ in configuration and material from sensor electrodes 940 on the front side of substrate 933. This asymmetry in the design of sensor device 930 enables sensor electrodes 940 (for measuring ion current in trap 938) and substrate contacts 944 (for connecting to PCB contacts) to be designed to suit their respective functions.

[0164] Figure 5 An embodiment of a sensor electrode 140 on the front side of a substrate 133 is shown, similar to Figure 4 The embodiment shown. Figure 5 Only the upper half of the base plate 133 is shown (the front side is shown).

[0165] In this embodiment, sensor electrode 140 is located in the center of through-hole 142, which includes a copper cylindrical coating 146 filled with adhesive 148. However, through-hole 142 does not have a copper cap. Instead, the front side of substrate 133 surrounding through-hole 142 is recessed, and titanium base layer 152 of sensor electrode 140 is formed within and around the recess. Platinum coating 154 completely covers titanium base layer 152 and thus conforms to the contours of the recess.

[0166] Figure 6 An embodiment of a sensor device 230 is shown, similar to Figure 4 The embodiment shown. Figure 6 The configuration of the sensor device 230 shown is similar to Figure 4 The sensor devices shown are very similar. Figure 6 The illustrated sensor device 230 includes a through-hole 242 extending through a substrate 233. The through-hole 242 includes a copper cylindrical coating 246 filled with an adhesive 248, but the through-hole 242 does not have a copper cap.

[0167] Figure 6 The sensor device 230 is shown with Figure 4 Another difference between the sensor devices shown is the shape of the sensor electrodes 240 on the front side of the substrate 233. Figure 6 In the illustrated sensor device 230 , the shape of the sensor electrode 240 is non-circular, and is generally an ellipse with straight sides.

[0168] In addition, the area of ​​the sensor electrodes 240 at the base of the wells 238 is offset from their corresponding through-holes 242. Therefore, each well 238 is offset from its corresponding through-hole 242. The shape and position of the sensor electrodes 240 will be referred to as Figure 7 Describe in more detail.

[0169] Figure 6 The sensor device 230 is shown with Figure 4Another major difference between the illustrated sensor devices is that a second PBO insulating layer 266 is provided over the edges of the first PBO insulating layer 264 and substrate contact 244 (leaving an area of ​​substrate contact 244 exposed for electrical contact with, for example, a solder ball).

[0170] Figure 7 Shown Figure 6 A plan view of sensor electrode 240 is shown. The relative positioning (and offset) of sensor electrode 240 on substrate 233, through-hole 242, and well 238 can be seen. Overall, sensor electrode 240 has a substantially straight-sided oval shape; however, platinum coating 254 does not have the exact same shape as titanium base layer 252. Near (below and around) well 238, platinum coating 254 extends beyond the boundaries of titanium base layer 252. Near (above and around) through-hole 242, platinum coating 254 does not extend to the boundaries of titanium base layer 252.

[0171] Figure 8 An embodiment of a sensor electrode 340 on the front side of a substrate 333 is shown, similar to Figure 6 and Figure 7 The embodiment shown. Figure 8 Only the upper half of the base plate 333 is shown (the front side is shown).

[0172] In this embodiment, sensor electrode 340 is offset from through-hole 342, which includes a copper-like barrel coating 346 filled with adhesive 348. However, through-hole 342 lacks a copper cap. Instead, the front side of substrate 333 surrounding through-hole 342 is recessed, and titanium base layer 352 of sensor electrode 340 is formed within and around the recess. Platinum coating 354 (over which the well of the sensing element will be located) does not completely cover titanium base layer 352, but rather is disposed only on a portion of titanium base layer 352, offset from and not covering titanium base layer 352 above through-hole 342.

[0173] Figure 9 A cross section of a sensor device 70 according to another embodiment of the present invention is schematically shown. The sensor device 70 may be used, for example, as a nanopore sensor, for example similar to Figure 3 The sensor device shown.

[0174] and Figure 3 and Figure 4 Similar to the sensor device shown, sensor device 70 includes a glass substrate 73 having formed thereon a photoresist support structure 74 having a plurality of walls 76 defining therebetween a plurality of wells 78. Support structure 74 is configured to support a nanoporous membrane above each well 78.

[0175] A sensor electrode 80 is formed on the front side of the substrate 73 at the bottom of each well 78. A plurality of vias 82 are formed through the substrate 73 to connect a corresponding plurality of sensor electrodes 80 to a corresponding plurality of substrate contacts 84 on the back side of the substrate 73.

[0176] Each of the through holes 82 through the substrate 73 includes a copper cylindrical coating 86 filled with an adhesive 88 to seal the through hole 82. The through holes 82 each have a diameter of approximately 70 μm. Figure 7 In the embodiment shown, the pitch between vias 82 (and the spacing between the plurality of substrate contacts 84 ) is approximately 800 μm, while the pitch between wells 78 is approximately 200 μm.

[0177] The sensor electrodes 80 on the front side of the substrate 73 are formed from two layers: a base layer 92 made of titanium (which is arranged to route from the through-holes 82 to the corresponding wells 78 to map the through-holes 82 pitch of approximately 800 μm to the pitch of the sensor electrodes 80 of approximately 200 μm) and a coating layer 94 made of platinum.

[0178] The through-holes 82 are offset from the center of the corresponding well 78 (and are not coaxial). Each sensor electrode 80 extends between the through-hole 82 and the corresponding offset well 78. The platinum coating 94 on each sensor electrode 80 does not cover the entire titanium base layer 92, but at least partially (e.g., completely) covers the bottom of the well 78 and the surrounding area of ​​the titanium base layer 92 below the photoresist support structure 74. Therefore, in some embodiments (which are configured similar to Figure 9 Unlike the example shown in FIG, the fluid in the well may be exposed to the substrate, such as around the edge of the sensor electrode 80. The platinum coating 94 does not extend to cover the portion of the titanium base layer 92 that contacts the through-hole 82.

[0179] The substrate contacts 84 on the back side of the substrate 73 are each formed of two or three layers: a base layer 96 made of copper or titanium, an optional intermediate layer 98 made of palladium, and a coating 102 made of gold. The intermediate layer 98 and the coating 102 can be applied to the base layer 96 using an electroless palladium immersion gold (EPIG) process. The copper or titanium base layer 96 is connected to the substrate 73 and extends over and around the cylindrical coating 86 in the through hole 82.

[0180] The substrate contacts 84 may be located at the center of their corresponding through holes 82 (e.g., Figure 7 The substrate contacts 84 on the right side of the substrate may be offset from their corresponding through holes 82 (as shown in FIG. Figure 7 (shown as substrate contacts 84 on the left).

[0181] The substrate contacts 84 on the rear side of the substrate 73 are configured for connection to input contacts of the PCB via corresponding spring contacts.

[0182] Figure 10 An embodiment of substrate contacts 184 located on the rear side of substrate 173 is shown, similar to Figure 9 The embodiment shown. Figure 8 Only the lower half of the base plate 173 is shown (the rear side is shown).

[0183] In this embodiment, the copper or titanium base layer 196 of the substrate contact 184 protrudes in the center of the substrate contact 184 (near the through-hole 182) and thus protrudes from the surrounding area of ​​the substrate contact 184. The gold coating 202 of the substrate contact 184 extends over the entire area of ​​the substrate contact 184.

[0184] The bumps on the substrate contacts 184 help engage corresponding spring contacts on the PCB, thereby helping to form a good electrical connection.

Claims

1. A sensor device for a nanopore sensor, the sensor device comprising: insulating substrate; one or more wells for containing a fluid; wherein the one or more wells are formed on a first side of the substrate; a sensor electrode for detecting ion current in each of the one or more wells; wherein the sensor electrodes are formed on the first side of the substrate at the base of the one or more wells; one or more through-holes extending through the substrate; wherein the one or more through-holes are connected to sensor electrodes; The one or more through-holes each include a conductive cap at one or both ends of the through-hole. 2 . The sensor device of claim 1 , wherein the one or more through-holes each include a conductive cap at both ends of the through-hole.

3. The sensor device according to claim 1 or 2, wherein the electrically conductive cover comprises a transition metal, such as copper.

4. A sensor device according to claim 1, 2 or 3, wherein the one or more through-holes are each filled with a conductive material. 5 . The sensor device according to claim 4 , wherein the conductive cover is formed of the same material as the conductive material filling the through-hole.

6. The sensor device of claim 1 , 2 or 3 , wherein the one or more through holes each comprise: a conductor extending through the through-hole; as well as a material arranged to retain the conductor in the through-hole; wherein the material arranged to retain the conductor in the through-hole is different from the material of the conductor. The sensor device according to claim 6 , wherein the conductor is connected to the conductive cover at one or both ends of the through hole.

8. The sensor device according to claim 6 or 7, wherein the conductive cover is formed of the same material as the material of the conductor.

9. A sensor device according to claim 6, 7 or 8, wherein the material arranged to retain the conductor in the through-hole substantially fills the through-hole.

10. A sensor device according to any one of claims 6 to 9, wherein the material arranged to retain the conductor in the through-hole comprises a sealant, such as an adhesive.

11. The sensor apparatus of any one of claims 6 to 10, wherein the conductor comprises a conductive cylinder at least partially lining the through-hole.

12. The sensor device of claim 11 , wherein the through-hole is defined by a wall passing through the substrate; and The conductive tube is at least partially lined with the wall of the through hole.

13. A sensor device according to claim 11 or 12, wherein the material arranged to retain the conductor in the through-hole substantially fills the conductive cylinder.

14. A sensor device according to claim 11, 12 or 13, wherein the material arranged to retain the conductor in the through-hole is arranged to substantially seal the conductive cylinder in the through-hole.

15. The sensor device of any preceding claim, wherein each of the one or more through-holes comprises a conductive cover proximate the first side of the substrate; The sensor electrodes are formed on corresponding conductive covers. 16 . The sensor device of claim 1 , wherein each of the sensor electrodes comprises an electrode base layer proximate to the substrate and an electrode coating exposed to the well. The sensor device according to claim 16 , wherein the electrode base layer is formed on the conductive cover.

18. The sensor device according to any one of the preceding claims, wherein the sensor device comprises: one or more contacts formed on the second side of the substrate; The one or more through-holes connect the sensor electrodes to the one or more contacts.

19. The sensor device of claim 18, wherein each of the one or more through-holes comprises a conductive cover proximate the second side of the substrate; The one or more contacts are formed on corresponding conductive covers.

20. The sensor device of any preceding claim, wherein the insulating substrate comprises a glass substrate.

21. A sensor device according to any preceding claim, wherein each well supports a membrane containing a nanopore channel inserted therein.

22. The sensor device of claim 21, wherein the membrane comprises amphiphilic molecules, such as lipids or polymers.

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