Devices with field effect transistors

Abstract

To provide devices which provide scalability, improved sensitivity and reduced noise for sequencing a polynucleotide.SOLUTION: A device comprises: a middle well comprising a fluidic tunnel; a cis well associated with a cis electrode, where a first nanoscale opening is disposed between the cis well and the middle well; a trans well associated with a trans electrode, where a second nanoscale opening is disposed between the trans well and the middle well; and a field effect transistor positioned between the first nanoscale opening and the second nanoscale opening. The FET comprises a source, a drain, and a channel. The channel comprises a gate oxide layer having an upper surface fluidically exposed to the middle well. The middle well fluidically connects the cis well to the trans well. The fluidic tunnel extends through the channel.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 047743, filed July 2, 2020, and U.S. Provisional Patent Application No. 63 / 200868, filed March 31, 2021, the contents of each of which are incorporated by reference in their entirety. [Background technology]

[0002] Various polynucleotide sequencing techniques involve carrying out large numbers of controlled reactions on a support surface or within predefined reaction chambers, which can then be observed or detected, and subsequent analysis can help identify the nature of the polynucleotides involved in the reactions.

[0003] Some of these polynucleotide sequencing techniques utilize nanopores that can provide a pathway for ionic current. For example, as a polynucleotide traverses through the nanopore, it affects the current flowing through the nanopore. Each passing nucleotide, or series of nucleotides that pass through the nanopore, results in a characteristic current. These characteristic currents of the traversing polynucleotide can be recorded to determine the sequence of the polynucleotide.

[0004] FIG. 1A shows a prior art nanopore sequencing device 1110 as shown in PCT Publication WO2019 / 160925. The prior art nanopore sequencing device 1110 includes a cis well 1114 associated with a cis electrode 1130, a trans well 1116 associated with a trans electrode 1134, and a field effect transistor (FET) 1122 positioned between the cis well 1114 and the trans well 1116. The FET 1122 includes a source 1150, a drain 1152, and a channel 1154. Below the cis well 1114 is a first cavity 1115 facing the cis well 1114. The trans well 1116 includes a second cavity 1117. A fluid tunnel 1121 extends through the FET 1122 from the first cavity 1115 to the trans well 1116. An electrolyte 1120 is disposed within the ciswell 1114, the first cavity 1115, and the transwell 1116.

[0005] Between the syswell 1114 and the first cavity 1115 is a nanopore 1118 disposed in the membrane 1124. The nanopore 1118 has a first nanoscale opening 1123 that fluidly and electrically connects the electrolyte from the syswell 1114 to the first cavity 1115. The first nanoscale opening 1123 has an inner diameter 1123'. As the polynucleotide 1129 traverses through the first nanoscale opening 1123, the sequence of the polynucleotide can be determined by measuring a change in the voltage of the FET sensor 1122. A second nanoscale opening 1125 in the base substrate 1162' fluidly connects the fluid tunnel 1121 and the second cavity 1117, the second nanoscale opening 1125 having an inner diameter 1125'.

[0006] Metal interconnects 1164' and 1166' are in electrical communication with source 1150 and drain 1152 of FET 1122. A relatively thick interlayer dielectric 1168, generally thicker than about 50 nm, surrounds channel 1154 and the top and bottom surfaces of FET sensor 1122 to form fluid tunnel 1121. FET sensor 1122 is in electrical communication with electrolyte 1120 at boundary 1156 where channel 1154 is closest to fluid tunnel 1121. As shown, the thickness of interlayer dielectric 1168 above or below channel 1154 can be about three times or more the thickness of channel 1154 of FET 1122. Summary of the Invention

[0007] Provided in the examples herein are devices for sequencing polynucleotides and methods of using the devices. One example of such a device is a nanopore device. In particular, examples include devices having field effect transistor (FET) sensors and porous structures.

[0008] The systems, devices, kits, and methods disclosed herein each have various aspects, no one of which is solely responsible for their desirable attributes. Without limiting the scope of the claims, some prominent features will now be briefly discussed. Numerous other embodiments are also contemplated, including embodiments having fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may also be arranged and ordered differently. After considering this discussion, and especially after reading the section entitled "Description of the Preferred Embodiments," one will appreciate how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

[0009] One example is a device comprising a central well with a fluid tunnel, a cis well associated with a cis electrode, with a first nanoscale opening disposed between the cis well and the central well, a trans well associated with a trans electrode, with a second nanoscale opening disposed between the trans well and the central well, and a field effect transistor (FET) positioned between the first nanoscale opening and the second nanoscale opening. In this example, the FET comprises a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having an upper surface fluidically exposed to the central well, and the central well fluidically connecting the cis well to the trans well. In some embodiments, the fluid tunnel extends through the channel. In alternative embodiments, the fluid tunnel is offset from (i.e., does not extend through) the FET channel.

[0010] Another example is a device comprising a central well with a fluid tunnel, a cis well associated with a cis electrode, with a first nanoscale opening disposed between the cis well and the central well, a cis well associated with a trans electrode, with a second nanoscale opening disposed between the trans well and the central well, and a field effect transistor (FET) positioned between the first nanoscale opening and the second nanoscale opening, the FET comprising a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having a top surface and a bottom surface, the surfaces being fluidically exposed to the central well, and the central well fluidically connecting the cis well to the trans well. In some embodiments, the fluid tunnel extends through the channel. In alternative embodiments, the fluid tunnel is offset from (i.e., does not extend through) the FET channel.

[0011] Yet another example is a device comprising a central well with a fluid tunnel, a cis well associated with a cis electrode, with a first nanoscale opening disposed between the cis well and the central well, a cis well associated with a trans electrode, with a porous structure disposed between the trans well and the central well, and a field effect transistor (FET) positioned between the trans well and the first nanoscale opening and the porous structure, the FET comprising a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having an upper surface fluidically exposed to the central well, and the central well fluidically connecting the cis well to the trans well. In some embodiments, the fluid tunnel extends through the FET channel. In alternative embodiments, the fluid tunnel is offset from (i.e., does not extend through) the FET channel.

[0012] Yet another embodiment is a method of using any of the aforementioned devices, comprising introducing an electrolyte into each of the cis well, trans well, central well, and fluidic tunnels of the device, and applying a voltage bias between the cis and trans electrodes, such that the electrical resistance of the first nanoscale opening changes in response to the identity of the base in the polynucleotide at the first nanoscale opening, and the potential of the electrolyte in the fluidic tunnel (V M ) which varies in response to a variation in electrical resistance of the first nanoscale opening; and measuring a response of the FET as a function of the base in the polynucleotide at the first nanoscale opening to identify the base in the polynucleotide.

[0013] It will be understood that any features of the devices and / or arrays disclosed herein may be combined together in any desired manner and / or configuration. It will further be understood that any features of the methods of using the devices may be combined together in any desired manner. It will further be understood that any combination of features of the methods and / or devices and / or arrays may be used together and / or combined with any of the embodiments disclosed herein. It will further be understood that any feature or combination of features of any of the devices and / or arrays and / or methods may be combined together in any desired manner and / or combined with any of the embodiments disclosed herein.

[0014] It is to be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below are considered to be part of the inventive subject matter disclosed herein and may be used to realize the benefits and advantages described herein. [Brief description of the drawings]

[0015] Features of examples of the present disclosure will become apparent upon reference to the following detailed description and the drawings in which like reference numbers correspond to similar, but perhaps not identical, components. For the sake of brevity, reference numbers or features having previously described functions may or may not be described in conjunction with the other drawings in which they appear. [Figure 1A] FIG. 1 is a cross-sectional side view of a prior art nanopore sequencing device. [Figure 1B] FIG. 1B shows a schematic circuit diagram of the electrical resistance provided by the prior art nanopore sequencing device of FIG. 1A. [Figure 2A] FIG. 1 is a cross-sectional side view of a nanopore sequencing device according to one embodiment. [Figure 2B-1] FIG. 3B is a cross-sectional top view of the nanopore sequencing device of FIG. 2A taken on line 3-3. [Figure 2B-2]FIG. 2B is a cross-sectional top view of the nanopore sequencing device of FIG. 2A taken on line 3'-3'. [Figure 3A] 1 shows a cross-sectional side view of an alternative embodiment of a nanopore sequencing device according to one embodiment. [Figure 3B] FIG. 3B is a cross-sectional top view of the nanopore sequencing device and FET sensor of FIG. 3A taken on line 3-3. [Figure 3C] 3B is a cross-sectional top view of the nanopore sequencing device and FET sensor of FIG. 3A taken on line 3'-3'. [Figure 3D] FIG. 3B is an alternative embodiment of a cross-sectional top view taken on line 3-3 of a nanopore sequencing device similar to FIG. 3A but with a wider embodiment of a FET sensor. [Figure 3E] FIG. 3B is an alternative embodiment of a cross-sectional top view taken on line 3'-3' of a nanopore sequencing device similar to FIG. 3A but with a wider embodiment of a FET sensor. [Figure 4A] FIG. 2 is another cross-sectional side view of an alternative embodiment of a nanopore sequencing device. [Figure 4B-1] FIG. 4B is a cross-sectional top view of the nanopore sequencing device of FIG. 4A taken on line 3-3. [Figure 4B-2] FIG. 4B is a cross-sectional top view of the nanopore sequencing device of FIG. 4A taken on line 3'-3'. [Figure 5A] FIG. 13 is a cross-sectional side view of yet another alternative embodiment of a nanopore sequencing device. [Figure 5B-1] FIG. 5B is a cross-sectional top view of the nanopore sequencing device of FIG. 5A taken on line 3-3. [Figure 5B-2] FIG. 5B is a cross-sectional top view of the nanopore sequencing device of FIG. 5A taken on line 3'-3'. [Figure 6] 1 is a cross-sectional side view of another illustrative alternative embodiment of a nanopore sequencing device. [Figure 7A] FIG. 13 is a cross-sectional side view of yet another illustrative alternative embodiment of a nanopore sequencing device having offset openings. [Figure 7B-1]FIG. 5B is a cross-sectional top view taken on line 3-3 of the nanopore sequencing device of FIG. 5A showing the offset openings. [Figure 7B-2] 5B is a cross-sectional top view taken at line 3'-3' of the nanopore sequencing device of FIG. 5A showing the offset openings. [Figure 8] 13A-13C are cross-sectional side views of further illustrative alternative embodiments of nanopore sequencing devices having vertical field effect transistors. [Figure 9] FIG. 13 is a cross-sectional side view of yet another alternative exemplary embodiment of a nanopore sequencing device having a field effect transistor with non-Faradadic metal electrodes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] All patents, applications, published applications, and other publications mentioned herein are incorporated herein by reference for the material referenced and in their entirety. If a term or phrase is used herein in a manner that is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the use herein takes precedence over the definition incorporated herein by reference.

[0017] One embodiment relates to a sequencing device that includes a field effect transistor (FET) sensor having a channel disposed between a source and a drain of the FET sensor. In many examples herein, the sequencing device is described as a nanopore device, but the device need not be a nanopore device and other configurations are possible. In one embodiment, the channel has a top surface, a bottom surface, or both exposed to an electrolyte in the device. The exposed top and / or bottom surfaces of the FET sensor provide an increased surface area of ​​the FET in electrical contact with the electrolyte that improves the sensitivity of the nanopore sequencing device. Furthermore, it has been found that increasing the surface area of ​​the FET exposed to the electrolyte reduces background electrical noise in the sensor and thus provides a multifactorial enhancement in the signal-to-noise ratio (SNR) when measuring a nucleic acid sequence in contact with a nanopore.

[0018] In one example, the nanopore sequencing system utilizes a FET sensor constructed with gate-all-around (GAA) transistors to further increase the signal-to-noise ratio of the device. This GAA technology allows the FET sensor to not only have a top surface exposed to the electrolyte, but also a bottom surface exposed to the electrolyte. More information regarding this structure is described below with reference to FIG. 4A. In one embodiment, one or more all-around gate transistors of the nanopore sequencing system may comprise top and bottom surfaces of source-drain channels exposed to the electrolyte, as shown in FIGS. 4A, 4B, and 4B'. In another embodiment, one or more all-around gate transistors of the nanopore sequencing system may comprise top and bottom surfaces of a plurality of source-drain channels exposed to the electrolyte, as shown in FIG. 6. In yet another embodiment, one or more all-around gate transistors of the nanopore sequencing system may comprise a vertical transistor as shown in FIG. 8.

[0019] In another embodiment, the FET is not in direct contact with the electrolyte. Instead, a non-Faraday metal electrode, as shown in Figure 9, is exposed to the electrolyte and transmits the detected signal to the sensing FET. This configuration significantly simplifies the manufacturing process and allows for better compatibility with conventional semiconductor process flows.

[0020] In another embodiment, the solid-state nanopore structure may be replaced with a porous structure, as discussed in more detail below, which may be more easily integrated into semiconductor manufacturing process flows.

[0021] As used herein, the term "exposed to the electrolyte" does not necessarily mean that a component is in direct contact with the electrolyte. For example, a FET sensor or a channel of a FET sensor exposed to the electrolyte may include a relatively thin layer of insulator between the sensor or channel and the electrolyte. For example, in one embodiment, the channel portion of a FET sensor located between the source and drain may be covered with a relatively thin layer of gate oxide, e.g., a thermally grown silicon dioxide layer, and the channel with that gate oxide is said to be "exposed to the electrolyte." Alternatively, the thin layer of insulator may be HfO2, Al2O3, silicon oxynitride, Si3N4, TiO2, Ta2O5, Y2O3, La2O3, ZrO2, ZrSiO4, barium strontium titanate, lead zirconate titanate, ZrSi x O y , or ZrAl x O y The gate oxide layer may be formed of a high-k dielectric such as SiO 2 , SiO 2 , or SiO 2 . The gate oxide layer may be about 10 nm thick, or in other embodiments less than about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 nm thick and still be within the embodiments described herein.

[0022] Electrical operation of nanopore sequencing devices Referring now to FIG. 1B, an equivalent circuit diagram of a nanopore device, such as the nanopore devices illustrated in FIGS. 2-7, is shown. An electrolyte is introduced into each of the cis well, trans well, central well, and fluid tunnel. A voltage difference V is applied between the cis electrode and the trans electrode. In some examples, a polynucleotide is driven through a first nanoscale opening of a first nanopore, for example, a protein nanopore. In alternative examples, the polynucleotide does not pass through the first nanopore, but a tagged nucleotide is incorporated by a polymerase acting on the polynucleotide. In certain embodiments, a single-stranded polynucleotide, a double-stranded polynucleotide, a tag or label of an incorporated nucleotide base, or other representative of an incorporated nucleotide base, as well as any combination thereof, may pass through the first nanopore. In certain embodiments, a tag or label of an incorporated nucleotide base may be separated or dissociated from the polynucleotide, and such a tag or label may pass through the first nanopore with or without the polynucleotide passing through the first nanopore. Examples are not limited to how a polynucleotide communicates with a nanopore to cause signal generation in a nanopore sequencing device. protein changes in response to the identity of the base at the first nanoscale opening, e.g., different tags of the tagged nucleotides change the resistance of the first nanoscale opening while a base of a polynucleotide passes through the first nanoscale opening or while the tagged nucleotide is incorporated by a polymerase acting on the polynucleotide.

[0023] In an embodiment, the second nanopore, e.g., the second nanoscale opening of the solid-state nanopore, has a fixed or substantially fixed electrical resistance R pore The potential of the electrolyte in the fluid tunnel, shown as voltage divider point M in FIG. 1C, is proportional to the electrical resistance R of the first nanoscale opening. proteinThus, measuring the response of the FET as a change in resistance at the first nanoscale opening allows for the determination of the resistance at the first nanoscale opening, and such information can be used to identify bases in a polynucleotide.

[0024] During nanopore sequencing operations, application of a potential (i.e., voltage difference V) across the first nanopore can force translocation of a nucleotide with an anion carrying charge through the first nanoscale opening. Depending on the bias, the nucleotide can be transported from the cis well to the central well or from the central well to the cis well. As the nucleotide passes through the first nanoscale opening, the current across the membrane 24 changes due to, for example, base-dependent blockage of the constriction. A signal from the change in current can be measured using a FET sensor. Examples of measuring the response of the FET include measuring the source-drain current or measuring the potential at the source and / or drain. Additionally, the resistance in the FET channel can be measured to identify the base at the first nanoscale opening.

[0025] In operation, the range of measured voltages may be selected from about -0.1V to about 0.1V or more, about -0.5V to about 0.5V or more, about -1V to about 1V or more, about -1.5V to about 1.5V or more, about -2.0V to about 2.0V or more, about -3.0V to about 3.0V or more, and about -5.0V to about 5.0V or more. Voltage polarity is typically applied such that the negatively charged nucleic acid is electrophoretically driven towards the trans electrode. In some cases, the voltage can be reduced or the polarity reversed to facilitate proper functioning of the device. In one non-limiting example, the resistance of the first nanoscale opening, R protein The resistance of the second nanoscale opening, R, can be about 0.5 to about 1 gigaohm (GΩ). pore may be approximately 50 megaohms (MΩ). protein varies as a function of the base of the polynucleotide at the first nanoscale opening.

[0026] The potential at the voltage divider point M is R protein and acts as the FET gate potential. The resistance R of a second nanoscale opening that may be formed in the solid-state nanopore pore is fixed, or at least substantially fixed, and is not regulated by the bases of the polynucleotide at the first nanoscale opening. For example, when the polynucleotide enters the constriction of the first nanoscale opening, the resistance R of the first nanoscale opening protein is adjusted based on the identity of the base in the polynucleotide. Alternatively, the resistance R of the first nanoscale opening protein is regulated based on the identity of the tag of the tagged nucleotide that is incorporated by the polymerase acting on the polynucleotide. protein can be relatively large, typically varying by 30-40% as a function of the different polynucleotide bases at the first nanoscale opening. protein can vary from about 0.001% to about 1%, from about 1% to about 5%, from about 5% to about 20%, from about 20% to about 40%, from about 40% to about 60%, or from 60% to about 100%. The resistance R of the second nanoscale opening, which can have a size larger than the first nanoscale opening, pore is R protein The function of the second nanoscale aperture is the fixed resistor R in the voltage divider. pore Since the purpose of the second nanoscale opening is to provide a current path (and not to read out a current associated with the first nanoscale opening), the second nanoscale opening may not need to be atomically precise.

[0027] The equivalent circuit shown in FIG. 1B is a voltage divider, with the potential at point M being the potential of the electrolyte in the fluid tunnel. This potential is the equivalent gate potential of the FET and establishes its operating point. The potential V M As the base identity of the polynucleotide changes, the current flowing through the FET (source-drain current) changes, providing a measure of the current flowing through the first nanoscale opening, and thus the identity of the polynucleotide base. In certain embodiments, the equivalent circuit of the nanopore device satisfies the following equation:

[0028] Potential V at point M M is given by V M =DV (1) During the ceremony,

[0029]

number

[0030] The signal that drives the FET sensor response is the potential V M Fluctuation of δV M From the above, the following relationships can be derived: δV M = VδD (3) where δD is the change in the voltage divider ratio upon changing the base of the polynucleotide at the first nanoscale opening.

[0031] signal δV M may exceed the limit of detection (LoD) of the FET sensor, i.e., VδD>LoD. Thus, the sensitivity of the nanopore device 10 improves as the LoD is reduced, V is increased, or δD is increased.

[0032] Therefore, the operating cis-trans bias V can satisfy:

[0033]

number

[0034] One example of a nanopore sequencing device having a FET sensor with increased surface area exposed to an electrolyte is shown in Figure 2A. Figure 2A is a side cross-sectional view of an exemplary device 10A. Figure 2B is a cross-sectional top view taken on line 3-3 in Figure 2A. Figure 2B' is a cross-sectional top view taken on line 3'-3' in Figure 2A.

[0035] The nanopore sequencing device 10A shown in Figures 2A, 2B, and 2B' includes a cis electrode 30A that connects to a cis well 14A. The cis well 14A has a lower portion that includes a first nanopore 18A disposed in a membrane 24A. The first nanopore 18A includes a first nanoscale opening 23A defined by the first nanopore 18A that communicates with a second nanoscale opening 25A disposed in a narrowed region 17A between the fluid tunnel 21A and the transwell 16A at the lower portion of the device 10A in the fluid tunnel 21A. As shown, the second nanoscale opening is formed in the substrate material 62A. The first nanopore 18A provides a fluidic path for electrolyte 20A to pass between the cis well 14A and the central well 15A. The fluid tunnel 21A provides a fluidic path for electrolyte to pass from the central well 15A through the second nanoscale opening 25A to the transwell 16A.

[0036] In one embodiment, the cis electrode 30A and the trans electrode 34A are at least substantially parallel to each other in an at least substantially horizontal direction. In other embodiments, the cis and trans electrodes can be in any suitable orientation relative to each other and to the nanopore device. The nanopore device 10A further includes a field effect transistor (FET) sensor 22A positioned between the first nanoscale opening 23A and the second nanoscale opening 25A. The FET sensor includes a source (S) 50A, a drain (D) 52A, and a channel 54A connecting the source 50A to the drain 52A. As shown in the top views, FIGS. 2B and 2B', the electrolyte 20A can be seen in the fluid tunnel 21A and extends through the channel 54A. Metal interconnects 64A and 66A are in electrical communication with the source 50A and drain 52A of the FET 22A through the etch stop layer 38A. Metal interconnects 64A and 66A communicate data from FET sensor 22A to a control system that monitors FET sensor 22A.

[0037] In the embodiment of nanopore device 10A shown in FIG. 2A, a thin layer of gate oxide 56A is grown around channel 54A such that its top surface 55A is fluidically exposed to electrolyte 20A in central well 15A. Gate oxide 56A may have a vertical surface fluidically exposed to electrolyte 20A in fluid tunnel 21A. The thin layer of gate oxide 56A separates channel 54A from electrolyte 20A and exposes channel 54A of FET sensor 22A to electrolyte 20A. The thickness of gate oxide 56A may be from about 1 to about 10 nm, or alternatively, from about 2 to about 4 nm. The thickness of gate oxide 56A is determined by the potential V M is selected to induce an inversion layer of electrons or holes that form a conductive path at the boundary of the channel 54A and the gate oxide 56A to provide measurable conduction between the source 50A and the drain 52A of the FET 22A.

[0038] In this configuration, the top surface 55A of the gate oxide 56A of the channel 54A fluidly exposes the channel 54A to the electrolyte in the central well 15A, as shown in FIG. 2B. By providing a large area of ​​the channel 54A exposed to the electrolyte 20A, the potential V M has better gate controllability over channel 54A.

[0039] Following equations (2) and (4) above, the expected level of separation can be calculated using the open pore resistance, R protein Assuming about 10%, an expected base splitter ratio D of about 0.1, then the variation δD is about 0.1×0.1=0.01. Using a FET sensor with 3 mV, the LoD means:

[0040]

number

[0041] Such a high cis-trans bias, V, may be incompatible with some choices of membrane 24A.

[0042] A reduction in LoD to about 0.2 mV reduces the required cis-trans bias V by about 15x to about 20 mV, which is compatible with typical membranes. This means that FET sensors with large gate areas would be advantageous. In a FET sensor such as that shown in prior art FIG. 1A, only a small portion of the channel 1154 is sensitive to voltage variations δV, mainly through the gate oxide 56A at the boundary of the fluid tunnel 21A. M In addition to exposing the channel 54A to variations in voltage through the boundaries of the fluid tunnel 21A, a structure having an exposed top surface 55A as shown in FIGS. 2A, 2B, and 2B′ is exposed to a voltage variation of δV M This significantly increases the sensitive area of ​​the FET exposed to the electrolyte 20A, improving the LoD, which scales as 1 / sqrt(A), where A is the area of ​​the channel 54A exposed to the electrolyte 20A.

[0043] The interlayer dielectric 68A can be any suitable insulator, including SiO2, HfO2, or Al2O3. When the interlayer dielectric 68A is silicon dioxide, etching can be performed to etch various components of the nanopore sequencing device. For example, etching can be performed using an etchant with high anisotropy, such as a fluorinated reactive ion etchant, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar, as some non-limiting examples.

[0044] Membrane 24A can be either a non-permeable or semi-permeable material. The first nanoscale opening 23A extends through membrane 24A. It should be understood that membrane 24A can be formed from any suitable natural or synthetic material, as described herein. In an embodiment, membrane 24A is selected from the group consisting of lipids and biomimetic equivalents of lipids. In a further example, membrane 24A is a synthetic membrane (e.g., a solid-state membrane, one example of which is silicon nitride), and the first nanoscale opening 23A is in a solid-state nanopore extending through membrane 24A. In an embodiment, the first nanoscale opening 23A extends through, for example, a polynucleotide nanopore, a polypeptide nanopore, or a solid-state nanopore, e.g., a carbon nanotube, disposed in the membrane.

[0045] In one embodiment, the source, drain, and channel of FET sensor 22A may be formed of silicon, and the surface of the silicon may be thermally oxidized to form a gate oxide on the channel of FET sensor 22A.

[0046] The first nanopore 18A can be any of a biological nanopore, such as a protein nanopore, a solid-state nanopore, a hybrid nanopore, such as a hybrid protein / solid-state nanopore, and a synthetic nanopore. In some embodiments, the nanopore has two open ends and a hollow core or hole (i.e., the first nanoscale opening) that connects the two open ends. When inserted into the membrane, one of the open ends of the nanopore faces the cis well and the other of the open ends of the nanopore faces the central well. In some cases, the open end of the nanopore that faces the central well can be fluidly connected to the fluid tunnel and aligned with at least a portion of the fluid tunnel. In other examples, the open end of the nanopore that faces the central well is fluidly connected to the fluid tunnel but is not aligned with the fluid tunnel. The hollow core of the nanopore allows for fluidic and electrical connection between the cis well and the central well. The diameter of the hollow core of the nanopore can range from about 1 nm up to about 1 μm and can vary along the length of the nanopore. In some embodiments, the open end facing the syswell can be larger than the open end facing the central well. In other embodiments, the open end facing the syswell can be smaller than the open end facing the central well.

[0047] The first nanopore 18A may insert directly into the membrane or the membrane may be formed around the nanopore. In an embodiment, the nanopore may insert itself into the formed lipid bilayer membrane. For example, the nanopore in its monomeric or multimeric form (e.g., octamer) may insert itself into the lipid bilayer and assemble into a transmembrane pore. In another embodiment, the nanopore may be added at a desired concentration to the ground side of the lipid bilayer where it inserts into the lipid bilayer. In yet another embodiment, the lipid bilayer may be formed across an aperture in a polytetrafluoroethylene (PTFE) film and positioned between the cis well and the central well. The nanopore may be added to the grounded cis compartment and may insert itself into the lipid bilayer in the area where the PTFE aperture is formed. In yet another embodiment, the nanopore may be tethered to a solid support (e.g., silicon, silicon oxide, quartz, indium tin oxide, gold, polymer, etc.). A tethering molecule, which may be part of the nanopore itself or may be attached to the nanopore, may attach the nanopore to a solid support. Attachment via a tethering molecule may be such that a single pore is immobilized (e.g., between a cis well and a central well). A lipid bilayer may then be formed around the nanopore 18. A lipid bilayer may then be formed around the nanopore.

[0048] In an embodiment, the inner diameter of the second nanoscale opening is at least about two times larger than the inner diameter of the first nanoscale opening. In another embodiment, the inner diameter of the second nanoscale opening is about three times larger than the inner diameter of the first nanoscale opening. In yet another embodiment, the inner diameter of the second nanoscale opening ranges from about two times larger than the inner diameter of the first nanoscale opening to about five times larger than the inner diameter of the first nanoscale opening. In an embodiment, the area of ​​the second nanoscale opening ranges from about five times to about ten times larger than the area of ​​the first nanoscale opening.

[0049] Further, in an embodiment, the first nanoscale opening inner diameter ranges from about 0.5 nm to about 3 nm, and the second nanoscale opening inner diameter 25A ranges from about 10 nm to about 20 nm. In another embodiment, the first nanoscale opening inner diameter 23A ranges from about 1 nm to about 2 nm, and the second nanoscale opening inner diameter 25A ranges from about 10 nm to about 20 nm. In yet another embodiment, the first nanoscale opening inner diameter 23A ranges from about 1 nm to about 3 nm, and the second nanoscale opening inner diameter 25A ranges from about 2 nm to about 20 nm. The exemplary ranges of the first nanoscale opening inner diameter 23A given above are intended to be the minimum diameter of the nanoscale opening 23A through the first nanopore 18A.

[0050] A substrate with an array of nanopore sequencing devices can have many different layouts of the first nanoscale openings on the array, including regular, repeating, and irregular patterns of nanoscale openings. In an embodiment, the first nanoscale openings can be arranged in a hexagonal lattice for close packing and improved density of the devices. Other array layouts can include, for example, rectilinear (i.e., rectangular) layouts, triangular layouts, and the like. As an example, the layout or pattern can be an xy format of the first nanoscale openings in rows and columns. In some other embodiments, the layout or pattern can be a repeating arrangement of the first nanoscale openings. In still other embodiments, the layout or pattern can be a random arrangement of the first nanoscale openings. The patterns can include spots, pillars, stripes, swirls, lines, triangles, rectangles, circles, arcs, checks, lattice patterns, diagonal lines, arrows, squares, and / or cross-hatches.

[0051] The layout of the nanoscale openings may be characterized in terms of the density of the first nanoscale openings (i.e., the number of first nanoscale openings in a defined area of ​​the substrate that comprises the array). For example, the array of first nanoscale openings may be 1 mm 2 Approximately 10 nanoscale openings per mm2 The density may be in the range of about 1,000,000 first nanoscale openings per mm. 2 At least about 10, 1 mm per 2 Approximately 5,000,1 mm per 2 Approximately 10,000,1mm per 2 Alternatively or additionally, the density may include a density of about 100,000 per mm 2 Approximately 1,000,000,1 mm per 2 Approximately 100,000 per mm 2 Approximately 10,000,1mm per 2 It should be further appreciated that the density of the first nanoscale openings in the substrate can be between one of the lower values ​​and one of the upper values ​​selected from the ranges above.

[0052] The layout of the first nanoscale openings in the array on the substrate may also be characterized in terms of the average pitch, i.e., the spacing from the center of a first nanoscale opening to the center of an adjacent first nanoscale opening (center-to-center spacing). The pattern may be regular such that the coefficient of variation around the average pitch is small, or the pattern may be irregular such that the coefficient of variation may be relatively large. In an embodiment, the average pitch may range from about 100 nm to about 500 μm. The average pitch may be, for example, at least about 100 nm, about 5 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the average pitch may be, for example, up to about 500 μm, about 100 μm, about 50 μm, about 10 μm, about 5 μm, or less. The average pitch for an exemplary array of devices may be between one of the lower values ​​and one of the upper values ​​selected from the ranges above. In an embodiment, the array may have a pitch (center-to-center spacing) of about 10 μm. In another embodiment, the array can have a pitch (center-to-center spacing) of about 5 μm, hi yet another embodiment, the array can have a pitch (center-to-center spacing) ranging from about 1 μm to about 10 μm.

[0053] As described above, a substrate for sequencing may include an array of nanopore sequencing devices. In one embodiment of a nanopore sequencing device, the transwell is fluidically connected to the ciswell by a central well and respective second and first nanoscale openings. In a substrate with an array of nanopore sequencing devices, one common ciswell and one common transwell may communicate with some or all of the nanopore sequencing devices in the array on the substrate. However, it should be understood that an array of nanopore devices may also include several ciswells that are fluidically isolated from each other or fluidically isolated from each other and fluidically connected to a respective one or more transwells defined in the substrate. Multiple ciswells may be desired, for example, to allow for the measurement of multiple polynucleotides on a single substrate. In some embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis electrode, one common trans electrode, one common cis well, one common trans well, and a plurality of nanopore sequencing devices such as that shown in FIG. 2A, where each nanopore sequencing device comprises a dual pore with a FET sensor and a first nanopore and a second nanopore. Each nanopore sequencing device of the plurality of nanopore sequencing devices can measure resistance or signal separately by its associated FET sensor. In other embodiments, each nanopore sequencing device can comprise a plurality of pores with three or more nanopores and FET sensors. In other embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis well, a plurality of trans wells, and a plurality of nanopore sequencing devices, where each nanopore sequencing device can be individually addressable with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of common cis wells, a plurality of trans wells, and a plurality of nanopore sequencing devices, each of which may be individually addressable with an individual trans electrode.

[0054] A cis-well of a nanopore sequencing device can be a fluid chamber defined by a sidewall connected to a substrate. In some examples, the sidewall and substrate can be integrally formed such that they are formed from a continuous piece of material (e.g., glass or plastic). In other examples, the sidewall and substrate can be separate components bonded to one another. In examples, the sidewall is a photopatternable polymer. In some examples, the cis-well is formed within a space defined by the cis-electrode, a portion of the substrate, and the membrane. The cis-well can have any suitable dimensions. In examples, the cis-well ranges from about 1 mm x 1 mm to about 3 cm x 3 cm. The cis-electrode, whose inner surface forms one surface of the cis-well, can be physically connected to the sidewall. The cis-electrode can be physically connected to the sidewall, for example, by an adhesive or another suitable fastening mechanism. The interface between the cis-electrode and the sidewall can seal a top portion of the cis-well.

[0055] A transwell of a nanopore sequencing device is a fluid chamber that may be defined in a portion of a substrate. The transwell may extend through the thickness of the substrate and may have openings at opposing ends of the substrate. In some examples, the transwell may have sidewalls defined by the substrate and / or by an interstitial region of the substrate, a lower surface defined by a trans electrode, and an upper surface defined by a base structure. Thus, a transwell may be formed within a space defined by the trans electrode, other portions of the substrate and / or the interstitial region, and the base structure. It should be understood that the upper surface of the transwell may include a second nanoscale opening to provide fluid communication to the central well. In some examples, the second nanoscale opening passes through the base structure. In some examples, the second nanoscale opening may be fluidly connected to and faces the narrowed region of the transwell.

[0056] A transwell can be a microwell (having at least one dimension on the micrometer scale, e.g., about 1 μm to about 1000 μm, but not including about 1000 μm) or a nanowell (having a maximum dimension on the nanoscale, e.g., about 10 nm to 1000 μm, but not including 1000 μm). A transwell can be characterized by its aspect ratio (e.g., in this example, width or diameter divided by depth or height). In an example, the aspect ratio of a transwell can range from about 1:1 to about 1:5. In another example, the aspect ratio of each transwell can range from about 1:10 to about 1:50. In an example, the aspect ratio of a transwell is about 3.3. The depth / height and width / diameter of a transwell can be selected to obtain a desired aspect ratio. The depth / height of each transwell can be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth can be up to about 1,000 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or less. The width / diameter of each transwell 16 can be at least about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the width / diameter can be up to about 1,000 μm, about 100 μm, about 10 μm, about 1 μm, about 0.5 μm, about 0.1 μm, about 50 nm, or less.

[0057] Syswells and transwells can be fabricated using a variety of techniques including, for example, photolithography, nanoimprint lithography, stamping techniques, embossing techniques, molding techniques, microetching techniques, etc. As will be appreciated by those skilled in the art, the technique used will depend on the composition and shape of the substrate and sidewalls. In embodiments, syswells can be defined by one or more sidewalls at the edge of a substrate, and transwells can be defined through the substrate.

[0058] The trans electrode, whose inner surface is the lower surface of the transwell, may be physically connected to the substrate. The trans electrode may be fabricated in the process of forming the substrate (e.g., during formation of the transwell). Microfabrication techniques that may be used to form the substrate and the trans electrode include lithography, metal deposition and lift-off, dry and / or spin-on film deposition, etching, etc. The interface between the trans electrode and the substrate may seal the lower portion of the transwell.

[0059] Examples of materials used to form the base structure 62A include silicon nitride (Si3N4), silicon carbide (SiC), aluminum oxide (Al2O3), hafnium oxide (HfO2), and tantalum pentoxide (Ta2O5). Examples of suitable deposition techniques for these materials include atomic layer deposition (ALD), as well as CVD. Examples of suitable material combinations for the base structure 62A include Si3N4, SiO2, SiC, or Al2O3.

[0060] The cis electrode used depends, at least in part, on the redox couple in the electrolyte. By way of example, the cis electrode can be gold (Au), platinum (Pt), carbon (C) (e.g., graphite, diamond, etc.), palladium (Pd), silver (Ag), copper (Cu), etc. In an embodiment, the cis electrode can be a silver / silver chloride (Ag / AgCl) electrode. In an embodiment, the cis well can maintain the electrolyte in contact with the first nanoscale opening. In some embodiments, the cis well can be in contact with the array of nanopores, and thus can maintain the electrolyte in contact with each of the nanopores in the array.

[0061] The transformer electrode used depends, at least in part, on the redox couple in the electrolyte. By way of example, the transformer electrode can be gold (Au), platinum (Pt), carbon (C) (e.g., graphite, diamond, etc.), palladium (Pd), silver (Ag), copper (Cu), etc. In an embodiment, the transformer electrode can be a silver / silver chloride (Ag / AgCl) electrode.

[0062] In some examples, for an Ag / AgCl electrode in NaCl or KCl solution, the relevant electrochemical half-reaction at the electrode is: Cis(cathode): AgCl+e - →Ag 0 +Cl - and Transformer (anode: Ag 0 +Cl - →AgCl+e - It is.

[0063] For every unit charge of current, one Cl atom is consumed at the transformer electrode. It should be understood that although the above discussion is in terms of an Ag / AgCl electrode in a NaCl or KCl solution, any electrode / electrolyte pair that can be used to pass current may apply.

[0064] In use, an electrolyte may be filled into the syswells, central wells, fluid tunnels, narrow regions, and transwells. In alternative embodiments, the electrolyte in the syswells, central wells, and transwells may be different. The electrolyte may be any electrolyte that can dissociate into counterions (cations and their associated anions). As an example, the electrolyte may be potassium cation (K + ) or sodium cation (Na +), which can dissociate into potassium cations and related anions. Electrolytes of this type include potassium cations and related anions, or sodium cations and related anions, or combinations thereof. Examples of potassium-containing electrolytes include potassium chloride (KCl), potassium ferricyanide (K3[Fe(CN)6]·3H2O or K4[Fe(CN)6]·3H2O), or other potassium-containing electrolytes (e.g., bicarbonates (KHCO3) or phosphates (e.g., KH2PO4, K2HPO4, K3PO4). Examples of sodium-containing electrolytes include sodium chloride (NaCl) or other sodium-containing electrolytes such as sodium bicarbonate (NaHCO3), sodium phosphates (e.g., NaH2PO4, Na2HPO4, or Na3PO4). As another example, the electrolyte can include ruthenium-containing cations (e.g., [Ru(NH3)6] 2+ or [Ru(NH3)6] 3+ The electrolyte can be any electrolyte that can dissociate into a lithium cation (Li + ), rubidium cation (Rb + ), magnesium cation (Mg + ), or calcium cation (Ca + ) may also be used.

[0065] In an example where multiple nanopore sequencing devices form an array on a substrate, each of the multiple nanopore sequencing devices in the array may share a common cis electrode and a common trans electrode. In another example, each of the multiple nanopore sequencing devices share a common cis electrode but have different trans electrodes. In yet another example, each of the multiple nanopore sequencing devices has a separate cis electrode and a separate trans electrode. In yet another example, each of the multiple nanopore sequencing devices has a separate cis electrode and shares a common trans electrode. When an array of nanopore devices is scaled, the volume of each transwell typically decreases dramatically as the cube of the well dimension (assuming a constant aspect ratio is maintained). In some examples, the array lifetime is about 48 hours or more, and the typical diameter of the transwell is about 100 μm or more.

[0066] Alternative Embodiments Figure 3A shows a variation of the device 10A shown in Figure 2A. As shown in Figure 3A, the nanopore sequencing device 10B includes similar components as the device shown in Figure 2A. However, the substrate material 62B shown in Figure 3A does not have the narrowed region as shown in Figure 2A. The substrate material 62B is more planar in configuration.

[0067] The nanopore sequencing device 10B shown in Figures 3A, 3B, and 3C includes a cis electrode 30B that connects to a cis well 14B. The cis well 14B has a lower portion that includes a first nanopore 18B disposed in a membrane 24B. The first nanopore 18B includes a first nanoscale opening 23B defined by the first nanopore 18B that communicates with a second nanoscale opening 25B between the fluid tunnel 21B and the transwell 16B at the lower portion of the device 10A in the fluid tunnel 21B. As shown, the second nanoscale opening 25B is formed in the substrate material 62B. The first nanopore 18B provides a fluidic path for the electrolyte 20B to pass between the cis well 14B and the central well 15B. The fluid tunnel 21B provides a fluidic path for the electrolyte to pass from the central well 15B through the second nanoscale opening 25B to the transwell 16B. In use, electrolytes may be filled in ciswell 14B, central well 15B, and transwell 16B. In alternative embodiments, the electrolytes in ciswell 14B, central well 15B, and transwell 16B may be different. In some embodiments, the diameter of first nanoscale opening 23B may be equal to or smaller than the opening of fluid tunnel 21B. A substrate for sequencing may include an array of nanopore sequencing devices. In one embodiment of a nanopore sequencing device, a transwell is fluidically connected to a ciswell by a central well and respective second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, one common ciswell and one common transwell may communicate with some or all of the nanopore sequencing devices in the array on the substrate. However, it should be understood that an array of nanopore devices may also include several ciswells fluidically isolated from each other or fluidically isolated from each other and fluidically connected to one or more transwells defined in the substrate. Multiple system wells may be desired, for example, to allow for the measurement of multiple polynucleotides on a single substrate.In some embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis electrode, one common trans electrode, one common cis well, one common trans well, and a plurality of nanopore sequencing devices such as that shown in FIG. 3A, where each nanopore sequencing device comprises a dual pore with a FET sensor and a first nanopore and a second nanopore. Each nanopore sequencing device of the plurality of nanopore sequencing devices can measure resistance or signal separately by its associated FET sensor. In other embodiments, each nanopore sequencing device can comprise a plurality of pores with three or more nanopores and FET sensors. In other embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis well, a plurality of trans wells, and a plurality of nanopore sequencing devices, where each nanopore sequencing device can be individually addressable with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of common ciswells, a plurality of transwells, and a plurality of nanopore sequencing devices, each of which may be individually addressable with an individual trans electrode.

[0068] In one embodiment, the cis electrode 30B and the trans electrode 34B are at least substantially parallel to each other in an at least substantially horizontal direction. In other embodiments, the cis and trans electrodes can be in any suitable orientation relative to each other and to the nanopore device. The nanopore device 10B further includes a field effect transistor (FET) sensor 22B positioned between the first nanoscale opening 23B and the second nanoscale opening 25B. The FET sensor includes a source (S) 50B, a drain (D) 52B, and a channel 54B connecting the source 50B to the drain 52B. As shown in the top views, FIGS. 3B and 3C, the electrolyte 20B can be seen in the fluid tunnel 21B and extends through the channel 54B. Metal interconnects 64B and 66B are in electrical communication with the source 50B and the drain 52B of the FET 22B through the etch stop layer 38B. Metal interconnects 64B and 66B communicate data from FET sensor 22B to a control system that monitors FET sensor 22B.

[0069] In the embodiment of nanopore device 10B shown in FIG. 3A, a thin layer of gate oxide 56B is grown around channel 54B such that its top surface 55B is fluidically exposed to central well 15B. Gate oxide 56B may have a vertical surface that is fluidically exposed to electrolyte 20B at fluid tunnel 21B. The thin layer of gate oxide separates channel 54B from electrolyte 20B and exposes channel 54B of FET sensor 22B to electrolyte 20B. In addition to exposing channel 54B to voltage fluctuations through gate oxide 56B at the boundary of fluid tunnel 21B, the structure with exposed top surface 55B, as shown in FIGS. 3A, 3B, and 3C, may also have a vertical surface that is fluidically exposed to electrolyte 20B at fluid tunnel 21B. M The thickness of gate oxide 56B can be from about 1 to about 10 nm thick, and in some embodiments, from about 2 to about 4 nm thick. The thickness of gate oxide 56B is determined by the potential V Mis selected such that a sufficiently strong electric field can be applied to induce an inversion layer of electrons or holes that form a conductive path at the channel 54B-gate oxide 56B boundary for conduction between the source 50B and the drain 52B.

[0070] The interlayer dielectric 68B can be any suitable insulator, including SiO2, HfO2, or Al2O3. When the interlayer dielectric 68B is silicon dioxide, etching can be performed to etch various components of the nanopore sequencing device. For example, etching can be performed using an etchant with high anisotropy, such as a fluorinated reactive ion etch, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar, as some non-limiting examples.

[0071] As shown, the transwell 16 in Figure 3A does not include a narrowed region as compared to Figure 2A. In some cases, this allows for a larger transwell 16B. The basic operating principles remain the same for the remainder of the nanopore sequencing device.

[0072] 3B and 3C are cross-sectional top views taken in FIG. 3A on lines 3-3 and 3'-3', respectively, and show an example of a FET sensor that is a nanowire transistor, ie, channel 54B has a nanowire configuration.

[0073] In a nanowire transistor, the channel 54B has a length along a direction from the source 50B to the drain 52B, a height along a direction from the cis electrode 30B to the trans electrode 34B, and a width along a direction at least partially or substantially perpendicular to both the length and the height. In one embodiment, the length can be at least about 10 times the width or height. The intersection between the fluid tunnel 21B and the channel 54B in the plane defined by the length and width can be disk-shaped, as shown in Figures 3B and 3C.

[0074] The LoD for a nanowire transistor having approximately 250 nm×20 nm×30 nm nanowires is approximately 3 mV, while the LoD for a nanowire transistor having approximately 10,000 nm×100 nm×30 nm wires is approximately 0.2 mV.

[0075] 3D and 3E are cross-sectional top views of the nanosheet FET sensor 22B' compared to the nanowire FET sensor 22B shown in FIGS. 3B and 3C. In the nanosheet FET sensor 22B', the channel 54B' has a nanosheet configuration. A thin layer of gate oxide 56B' separates the top surface of the channel 54B' from the electrolyte 20B' and exposes the channel 54B' of the FET sensor 22B' to the electrolyte 20B'. The thickness of the gate oxide 56B' can be about 1 to about 10 nm, preferably about 2 to about 4 nm. The thickness of the gate oxide 56B' is determined by the potential V M is selected such that a sufficiently strong electric field can be applied to induce an inversion layer of electrons or holes that form a conductive path at the channel 54B'-gate oxide 56B' boundary to conduct between the source 50B' and the drain 52B'. The sensing area of ​​the FET exposed to the electrolyte 20B' is significantly increased, thus further improving the LoD.

[0076] In the nanosheet FET sensor 22B', the channel 54B' has a length along the direction from the source 50B' to the drain 52B', a height along the direction from the cis electrode to the trans electrode, and a width along a direction at least partially or substantially perpendicular to both the length and the height. The length can be at least about twice the height, and the width can be at least about twice the height. In other embodiments, the length can be at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more times the height, and the width can be at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more times the height. The intersection between the fluid tunnel 21B' and the channel 54B' in the plane defined by the length and width can be elliptical as shown in Figures 3D and 3E (see, for example, the elliptical boundary of 56B').

[0077] Alternatively, the intersection between the fluid tunnel 21B' and the channel 54B' in the nanosheet transistor can be of almost arbitrary shape and size, potentially increasing the sensing area of ​​the FET even more and thus lowering the LoD even more. Because the size and shape requirements of the fluid tunnel can be relaxed, the manufacturability of the device can be improved.

[0078] Additional Examples Figures 4A, 4B, and 4B' illustrate another example of the nanopore device shown in Figures 2A, 2B, and 2B' that uses gate-all-around (GAA) transistors. Figure 4A is a cross-sectional side view of a nanopore sequencing device 10C. Figure 4B is a cross-sectional top view taken on line 3-3 in Figure 4A. Figure 4B' is a cross-sectional top view taken on line 3'-3' in Figure 4A.

[0079] The nanopore sequencing device 10C shown in Figures 4A, 4B, and 4B' includes a cis electrode 30C that connects to a cis well 14C. The cis well 14C has a lower portion that includes a first nanopore 18C disposed in a membrane 24C. The first nanopore 18C includes a first nanoscale opening 23C defined by the first nanopore 18C that communicates with a second nanoscale opening 25C disposed in a narrowed region 17C between the fluid tunnel 21C and the transwell 16C at the lower portion of the device 10C in the fluid tunnel 21C. As shown, the second nanoscale opening is formed in the substrate material 62C. The first nanopore 18C provides a fluidic pathway for the electrolyte 20C to pass between the cis well 14C and the central well 15C. The fluid tunnel 21C provides a fluidic pathway for the electrolyte to pass from the central well 15C through the second nanoscale opening 25C to the transwell 16C. The substrate for sequencing may include an array of nanopore sequencing devices. In one embodiment of a nanopore sequencing device, the transwell is fluidically connected to the ciswell by the central well and the respective second and first nanoscale openings. In a substrate with an array of nanopore sequencing devices, one common ciswell and one common transwell may be in communication with some or all of the nanopore sequencing devices in the array on the substrate. However, it should be understood that an array of nanopore devices may also include several ciswells that are fluidically isolated from each other or fluidically isolated from each other and fluidically connected to each one or more transwells defined in the substrate. Multiple ciswells may be desired, for example, to allow for the measurement of multiple polynucleotides on a single substrate. In some embodiments, a substrate having an array of nanopore sequencing devices comprises one common cis electrode, one common trans electrode, one common cis well, one common trans well, and a plurality of nanopore sequencing devices such as that shown in FIG. 4A, each nanopore sequencing device comprising a FET sensor and a dual pore having a first nanopore and a second nanopore.Each nanopore sequencing device of the multiple nanopore sequencing devices can measure the resistance or signal separately by its associated FET sensor. In other embodiments, each nanopore sequencing device can comprise multiple pores with three or more nanopores and FET sensors. In other embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis well, multiple trans wells, and multiple nanopore sequencing devices, each of which can be individually addressable with an individual trans electrode. In other embodiments, the substrate with the array of nanopore sequencing devices comprises multiple common cis wells, multiple trans wells, and multiple nanopore sequencing devices, each of which can be individually addressable with an individual trans electrode.

[0080] In one embodiment, the cis electrode 30C and the trans electrode 34C are at least substantially parallel to each other in an at least substantially horizontal direction. In other embodiments, the cis and trans electrodes can be in any suitable orientation relative to each other and to the nanopore device. The nanopore device 10C further includes a field effect transistor (FET) sensor 22C positioned between the first nanoscale opening 23C and the second nanoscale opening 25C. The FET sensor includes a source (S) 50C, a drain (D) 52C, and a channel 54C connecting the source 50C to the drain 52C. As shown in the top views, FIGS. 4B and 4B', the electrolyte 20C can be seen in the fluid tunnel 21C and extends through the channel 54C. Metal interconnects 66C and 64C are in electrical communication with the source 50C and drain 52C of the FET 22C through the etch stop layer 38C. The metal interconnects communicate data from the FET sensor 22C to a control system that monitors the FET sensor 22C.

[0081] In the nanopore sequencing device 10C shown in FIG. 4A, most of the material directly above the line 3-3 separating the channel 54C from the electrolyte 20C is removed, exposing the channel 54C of the FET sensor 22C to the electrolyte 20C. In addition, most of the material directly below the channel 54C is removed or hollowed out, exposing the channel 54C to the electrolyte from below as well, which may be formed by undercutting the active area 54C of the FET sensor 22C by known methods. Only a thin layer of gate oxide 56C is grown around the channel 54C. The top surface 55C and bottom surface 58C of the gate oxide 56C are fluidically exposed to the electrolyte 20C in the central well 15C and the fluidic channel 21C. The gate oxide 56C may have a vertical surface fluidically exposed to the electrolyte 20C in the fluidic tunnel 21C. A thin layer of gate oxide 56C separates the channel 54C from the electrolyte 20C and exposes the channel 54C of the FET sensor 22C to the electrolyte 20C. The thickness of the gate oxide 56C can be from about 1 to about 10 nm, and in some embodiments, from about 2 to about 4 nm. The thickness of the gate oxide 56C is determined by the potential V M is selected such that a sufficiently strong electric field can be applied to induce an inversion layer of electrons or holes that form a conductive path at the channel 54C-gate oxide 56C boundary for conduction between the source 50C and the drain 52C.

[0082] Such a configuration of the FET sensor 22C shown in FIG. 4A allows for exposure of a relatively large area of ​​the channel 54C to the electrolyte 20C (compared to FIG. 2A). Thus, the channel 54C uses an upper surface 55C and a lower surface 58C to fluidly connect the channel 54C to the central well 15C. Thus, the potential V M has advantageous gate controllability over channel 54C, further reducing LoD. In addition to exposing channel 54C to voltage fluctuations through gate oxide 56B at the boundaries of fluid tunnel 21C, the structure of FET sensor 22C with exposed top surface 55C and bottom surface 58C, as shown in FIGS. 4A, 4B, and 4B′, provides a high-level of sensitivity to δV MThe exposed sensing area of ​​the FET is significantly increased, improving LoD.

[0083] The interlayer dielectric 68C can be any suitable insulator, such as SiO2, HfO2, or Al2O3. When the interlayer dielectric 68C is silicon dioxide, etching can be performed to etch various components of the nanopore sequencing device. For example, etching can be performed using an etchant with high anisotropy, such as a fluorinated reactive ion etchant, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar, as some non-limiting examples.

[0084] The membrane 24C can be either a non-permeable or semi-permeable material. The first nanoscale opening 23C extends through the membrane 24C. It should be understood that the membrane 24C can be formed from any suitable natural or synthetic material, as described herein. In an embodiment, the membrane 24C is selected from the group consisting of lipids and biomimetic equivalents of lipids. In a further example, the membrane 24C is a synthetic membrane (e.g., a solid-state membrane, one example of which is silicon nitride), and the first nanoscale opening 23C is in a solid-state nanopore that extends through the membrane 24C. In an embodiment, the first nanoscale opening 23C extends through, for example, a polynucleotide nanopore, a polypeptide nanopore, or a solid-state nanopore, e.g., a carbon nanotube, disposed in the membrane.

[0085] In one embodiment, the source, drain, and channel of FET sensor 22C may be formed of silicon, and the surface of the silicon may be thermally oxidized to form a gate oxide on the channel of FET sensor 22C.

[0086] The first nanopore 18C can be any of a biological nanopore, a solid-state nanopore, a hybrid nanopore, and a synthetic nanopore. In some embodiments, the first nanopore 18C has two open ends and a hollow core or hole (i.e., the first nanoscale opening 23C) connecting the two open ends. When inserted into the membrane 24C, one of the open ends of the first nanopore 18C faces the cis well 14C, and the other of the open ends of the first nanopore 18C faces the central well 15C. In some cases, the open end of the first nanopore 18C facing the central well 15C can be fluidly connected to the fluid tunnel 21C and aligned with at least a portion of the fluid tunnel 21C. In other examples, the open end of the first nanopore 18C facing the central well 15C is fluidly connected to the fluid tunnel 21C but is not aligned with the fluid tunnel 21C. The hollow core of the first nanopore 18C allows for fluidic and electrical connection between the cis well 14C and the central well 15C. The diameter of the hollow core of the first nanopore 18C can range from about 1 nm up to about 1 μm and can vary along the length of the first nanopore 18C. In some embodiments, the open end facing the cis well 14C can be larger than the open end facing the central well 15C. In other embodiments, the open end facing the cis well 14C can be smaller than the open end facing the central well 15C.

[0087] Further Examples Figures 5A, 5B, and 5B' illustrate a modification of the nanopore device shown in Figures 2A, 2B, and 2B' that uses a porous structure 2500D in place of the second nanoscale opening 25A shown in Figure 2A. Figure 5A is a side cross-sectional view of the modified exemplary device 10D. Figure 5B is a cross-sectional top view taken on line 3-3 in Figure 5A. Figure 5B' is a cross-sectional top view taken on line 3'-3' in Figure 5A.

[0088] The nanopore sequencing device 10D shown in Figures 5A, 5B, and 5B' includes a cis electrode 30D that connects to a cis well 14D. The cis well 14D has a lower portion that includes a first nanopore 18D disposed in a membrane 24D. The first nanopore 18D includes a first nanoscale opening 23D defined by the first nanopore 18D that communicates with a fluid tunnel 21C to a narrowed region 17D of a transwell 16D at the lower portion of the device 10D. The first nanopore 18D provides a fluid path for electrolyte 20D to pass between the cis well 14D and the central well 15D. The fluid tunnel 21D provides a fluid path for electrolyte to pass from the central well 15D to the transwell 16D. A porous structure 2500D is disposed between the transwell 16D and the central well 15D. A substrate for sequencing may include an array of nanopore sequencing devices. In one example of a nanopore sequencing device, the transwell is fluidically connected to the ciswell by the central well and the respective second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, one common ciswell and one common transwell may be in communication with some or all of the nanopore sequencing devices in the array on the substrate. However, it should be understood that an array of nanopore devices may also include several ciswells that are fluidically isolated from each other, or fluidically isolated from each other and fluidically connected to one or more transwells defined in the substrate. Multiple ciswells may be desired, for example, to allow for the measurement of multiple polynucleotides on a single substrate. In some embodiments, a substrate having an array of nanopore sequencing devices includes one common cis electrode, one common trans electrode, one common cis well, one common trans well, and multiple nanopore sequencing devices, such as those shown in FIG. 5A, where each nanopore sequencing device includes a FET sensor and a dual pore with a first nanopore and a second nanopore. Each nanopore sequencing device of the multiple nanopore sequencing devices can separately measure the resistance or signal with its associated FET sensor.In other embodiments, each nanopore sequencing device may comprise a plurality of pores with three or more nanopores and FET sensors. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a common cis well, a plurality of trans wells, and a plurality of nanopore sequencing devices, each of which may be individually addressable with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of common cis wells, a plurality of trans wells, and a plurality of nanopore sequencing devices, each of which may be individually addressable with an individual trans electrode.

[0089] In one embodiment, the cis electrode 30D and the trans electrode 34D are at least substantially parallel to each other in an at least substantially horizontal direction. In other embodiments, the cis and trans electrodes can be in any suitable orientation relative to each other and to the nanopore device. The nanopore device 10D further includes a field effect transistor (FET) sensor 22D positioned between the first nanoscale opening 23D and the porous structure 2500D. The FET sensor includes a source (S) 50D, a drain (D) 52D, and a channel 54D connecting the source 50D to the drain 52D. As shown in the top views, FIGS. 5B and 5B', the electrolyte 20D can be seen in the fluid tunnel 21D and extends through the channel 54D. Metal interconnects 66D and 64D are in electrical communication with the source 50D and drain 52D of the FET 22D through the etch stop layer 38D. Metal interconnects 66D and 64D communicate data from FET sensor 22D to a control system that monitors FET sensor 22D.

[0090] In the embodiment of nanopore device 10D shown in FIG. 5A, a thin layer of gate oxide 56D is grown around channel 54D such that its top surface 55D is fluidically exposed to the central well. Gate oxide 56D can have a vertical surface fluidically exposed to electrolyte 20D in fluid tunnel 21D. The gate oxide separates channel 54D from electrolyte 20D and exposes channel 54D of FET sensor 22D to electrolyte 20D. The thickness of gate oxide 56D can be from about 1 to about 10 nm, and in some embodiments, from about 2 to about 4 nm. The thickness of gate oxide 56D is determined by the potential V M is selected such that a sufficiently strong electric field can be applied to induce an inversion layer of electrons or holes that form a conductive path at the channel 54D-gate oxide 56D boundary for conduction between the source 50D and the drain 52D.

[0091] In this configuration, channel 54D has an upper surface that fluidly connects channel 54D to the electrolyte in central well 15D, as shown in FIG. 5B. By increasing the area of ​​channel 54D exposed to electrolyte 20D, potential V M has better gate control over channel 54D. In addition to exposing channel 54D to voltage fluctuations through gate oxide 56D at the boundary of fluid tunnel 21D, the structure with the top surface of channel 54D exposed, as shown in FIGS. 5A, 5B, and 5B′, provides a gate controllability of δV M The exposed sensing area of ​​the FET is significantly increased, improving LoD.

[0092] In FIG. 2A, for example, the second nanoscale opening 25A formed in the solid-state nanopore defines part of the operation of the device. It can be a challenge to use current complementary metal-oxide-semiconductor (CMOS) technology to create nanopores smaller than about 10 nm. However, this choice limits the divider ratio D to about 0.1, thereby reducing the fluctuation δD when the base of the polynucleotide at the first nanoscale opening 23 changes, which in turn drives the required cis-trans bias V. In certain embodiments, the equivalent circuit of the nanopore device satisfies the following equation:

[0093] In the device 10A of FIG. 2A, the signal detected by the FET sensor is

[0094]

number

[0095] This signal is at a maximum when

[0096]

number

[0097] Fabrication of solid-state nanopores with similar size and resistance to protein nanopores remains challenging for current CMOS-based fabrication techniques. Furthermore, a single solid-state nanopore that meets this requirement may have variable resistance, as a polynucleotide, e.g., a single-stranded DNA polymer with a width of about 1 nm, would be expected to have a large change in resistance when traversing a solid-state nanopore with an opening of similar width.

[0098] In contrast, in FIG. 5A, the second nanoscale opening is replaced with a porous structure 2500D, such as a nanoporous frit or membrane. The structure and function of the frit is similar to that of the glass frit used in the reference electrode. The pores in the frit may be randomly distributed and may form complex pathways. The porosity of the frit is selected to be sufficient to establish electrical continuity across the frit (i.e., large enough for ionic species from the electrolyte to pass through), but small enough that a significant resistance to ionic current is established. 1 mm 2 The resistance of a typical frit having a size of is on the order of 1 MΩ. Thus, a 100 nm×100 nm frit can be expected to have a resistance of over 1 TΩ. A typical frit has a pore size of about a few nm and a thickness of about 1 mm. The porosity and thickness of the frit should be tailored to achieve the following desired targets: R frit =R protein (9)

[0099] There are many manufacturing compatible materials that can be used for frits, such as porous low-κ dielectrics (e.g., organosilicate glass (SiCOH) such as porous organosilicate glass (SiCOH)) that can be used and fabricated with porosity that can be tuned as high as 50%. Precursors with ring structures such as cyclomethicone, e.g., decamethylcyclopentasiloxane ([(CH3)2SiO]5), are sometimes used to achieve intrinsic porosity of a few percent. Porosity as high as 50% can be achieved from biphasic precursors such as a mixture of DMDS (dimethyl disulfide, CH3SSCH3) and α-terpenine, where the α-terpenine phase is removed via thermal treatment. The structure of the resulting material can vary from worm-like mesopores arranged in an irregular manner to well-ordered channel-like arrays with typical pore sizes on the order of a few nm. Regular porosity with a period on the order of tens of nm has also been demonstrated.

[0100] Additional Examples 6 is a cross-sectional, schematic, and partial cross-sectional view of yet another exemplary nanopore sequencing device 10E. FIG 6 illustrates a modification of FIG 5A in which the FET sensor uses a stack of channels 601 to further improve SNR and gate controllability.

[0101] The nanopore sequencing device 10E shown in FIG. 6 includes a cis electrode 30E that connects to a cis well 14E. The cis well 14E has a bottom portion that includes a first nanopore 18E disposed in a membrane 24E. The first nanopore 18E includes a first nanoscale opening 23E defined by the first nanopore 18E that communicates with a fluid tunnel 21E to a narrowed region 17E of a transwell 16E at the bottom portion of the device 10E. The first nanopore 18E provides a fluid path for electrolyte 20E to pass between the cis well 14E and the central well 15E. The fluid tunnel 21E provides a fluid path for electrolyte to pass from the central well 15E to the transwell 16E. A porous structure 2500E is disposed between the transwell 16E and the central well 15E. A substrate for sequencing may include an array of nanopore sequencing devices. In one example of a nanopore sequencing device, the transwell is fluidically connected to the ciswell by the central well and the respective second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, one common ciswell and one common transwell may be in communication with some or all of the nanopore sequencing devices in the array on the substrate. However, it should be understood that an array of nanopore devices may also include several ciswells that are fluidically isolated from each other, or fluidically isolated from each other and fluidically connected to a respective one or more transwells defined in the substrate. Multiple ciswells may be desired, for example, to allow for the measurement of multiple polynucleotides on a single substrate. In some embodiments, a substrate having an array of nanopore sequencing devices includes one common cis electrode, one common trans electrode, one common cis well, one common trans well, and multiple nanopore sequencing devices, such as those shown in FIG. 6, where each nanopore sequencing device includes a FET sensor and a dual pore with a first nanopore and a second nanopore. Each nanopore sequencing device of the multiple nanopore sequencing devices can separately measure the resistance or signal with its associated FET sensor.In other embodiments, each nanopore sequencing device may comprise a plurality of pores with three or more nanopores and FET sensors. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a common cis well, a plurality of trans wells, and a plurality of nanopore sequencing devices, each of which may be individually addressable with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of common cis wells, a plurality of trans wells, and a plurality of nanopore sequencing devices, each of which may be individually addressable with an individual trans electrode.

[0102] In one embodiment, the cis electrode 30E and the trans electrode 34E are at least substantially parallel to each other in an at least substantially horizontal direction. In other embodiments, the cis and trans electrodes can be in any suitable orientation relative to each other and to the nanopore device. The nanopore device 10D further includes a field effect transistor (FET) sensor 22E positioned between the first nanoscale opening 23E and the porous structure 2500E. The FET sensor includes a source (S) 50E and a drain (D) 52E. The metal interconnects 66E and 64E are in electrical communication with the source 50E and the drain 52E of the FET 22E through the etch stop layer 38E. The metal interconnects 66E and 64E communicate data from the FET sensor 22E to a control system that monitors the FET sensor 22E.

[0103] The FET sensor 22E is modified such that the FET further includes a stack of channels 601 aligned substantially horizontally and connecting the source 50E to the drain 52E. In the embodiment of the nanopore device 10E shown in FIG. 5E, a thin layer of gate oxide 56E is grown around the stack of channels 601. The thin layer of gate oxide separates the channels from the electrolyte 20E and exposes the channels of the FET sensor 22E to the electrolyte 20E. The thickness of the gate oxide 56E can be from about 1 to about 10 nm, and in some embodiments, from about 2 to about 4 nm. The thickness of the gate oxide 56E is selected such that an electric field strong enough to provide a potential VM can induce an inversion layer of electrons or holes at the channel 54E-gate oxide 56E interface that constitutes a conductive pathway for conduction between the source 50E and the drain 52E. Thus, each channel 605 of the plurality of channels has a top surface 607 and a bottom surface 608 of the gate oxide that fluidly connect to the central well 15E. Each channel 605 can have a vertical surface that fluidly connects to a fluid tunnel 21E. The fluid tunnel 21E extends through each of the multiple channels. Thus, the total FET sensing area can be increased by increasing the number of channels in the stack. By increasing the area of ​​the channel 601 exposed to the electrolyte 20E, the potential V M has better gate control over the channel. This configuration reduces δV M This significantly increases the exposed sensing area of ​​the FET, improving LoD.

[0104] The device 10E of FIG. 6 includes a porous structure 2500E, such as a nanoporous frit or membrane. However, it should be understood that this embodiment may also use a second nanoscale opening, similar to the structure of FIG. 2A. However, in the embodiment shown in FIG. 6, the structure and function of the frit is similar to that of a glass frit used in a reference electrode. The porosity of the frit is selected to be sufficient to establish electrical continuity across the frit (i.e., large enough for ionic species from the electrolyte to pass through), but small enough that diffusion of the polymer across it is not possible. 1 mm2 The resistance of a typical frit of this size is on the order of 1 MΩ, so a 100 nm×100 nm frit can be expected to have a resistance of over 1 TΩ. A typical frit has a pore size of about a few nm and a thickness of about 1 mm. Adjusting the porosity and thickness of the frit can be used to reduce R frit This should achieve the desired target.

[0105] Other aspects and advantages of the present disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the disclosure.

[0106] While only certain features of the embodiments have been illustrated and described herein, many modifications and changes will occur to those skilled in the art, and it is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes.

[0107] Various modifications and variations of the described methods and compositions will be apparent to those of skill in the art without departing from the scope of the embodiments described herein. It should be understood that the embodiments as claimed should not be unduly limited to the particular embodiments disclosed herein. Indeed, various modifications that are obvious to those skilled in the relevant arts are intended to be within the scope of the following claims.

[0108] Other aspects and advantages of the present disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the disclosure.

[0109] While only certain features have been illustrated and described herein, many modifications and changes will occur to those skilled in the art and it is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes.

[0110] Alternative Embodiments Figures 7A, 7B, and 7B' illustrate another variation of the nanopore device shown in Figures 2A, 2B, and 2B' having an alternative arrangement of fluid tunnels relative to the field effect transistors. Figure 7A is a cross-sectional side view of a nanopore sequencing device 10F. Figure 7B is a cross-sectional top view taken on line 3-3 in Figure 7A. Figure 7B' is a cross-sectional top view taken on line 3'-3' in Figure 7A.

[0111] The nanopore sequencing device 10F shown in Figures 7A, 7B, and 7B' includes a cis electrode 30F that connects to a cis well 14F. The cis well 14F has a bottom portion that includes a first nanopore 18F disposed in a membrane 24F. The first nanopore 18F includes a first nanoscale opening 23F defined by the first nanopore 18F that communicates with an offset fluid tunnel 21F to a second nanoscale opening 25F. The second nanoscale opening 25F is disposed in a narrowed region 17F between the offset fluid tunnel 21F and a transwell 16F at the bottom portion of the device 10F. As shown, the second nanoscale opening 25F is formed in a substrate material 62F. In other embodiments, the substrate material 62F does not have a narrowed region, similar to the structure shown in Figure 3A, but is more planar in configuration.

[0112] A first nanopore 18F provides a fluid pathway for electrolyte 20F to pass between ciswell 14F and central well 15F. As shown in Figure 7B, a fluid tunnel 21F is positioned offset from the central portion of the device and provides a fluid pathway for electrolyte to pass from central well 15F through a second nanoscale opening 25F to transwell 16F.

[0113] The substrate for sequencing may include an array of nanopore sequencing devices 10F. In one embodiment of a nanopore sequencing device, the transwell is fluidically connected to the ciswell by a central well and respective second and first nanoscale openings. In a substrate with an array of nanopore sequencing devices, one common ciswell and one common transwell may be in communication with some or all of the nanopore sequencing devices in the array on the substrate. However, it should be understood that an array of nanopore devices may also include several ciswells fluidically isolated from each other or fluidically isolated from each other and fluidically connected to a respective one or more transwells defined in the substrate. Multiple ciswells may be desired, for example, to allow for the measurement of multiple polynucleotides on a single substrate. In some embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis electrode, one common trans electrode, one common cis well, one common trans well, and a plurality of nanopore sequencing devices such as that shown in FIG. 7A, where each nanopore sequencing device comprises a dual pore with a FET sensor and a first nanopore and a second nanopore. Each nanopore sequencing device of the plurality of nanopore sequencing devices can measure resistance or signal separately by its associated FET sensor. In other embodiments, each nanopore sequencing device can comprise a plurality of pores with three or more nanopores and FET sensors. In other embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis well, a plurality of trans wells, and a plurality of nanopore sequencing devices, where each nanopore sequencing device can be individually addressable with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of common cis wells, a plurality of trans wells, and a plurality of nanopore sequencing devices, each of which may be individually addressable with an individual trans electrode.

[0114] In one example, the cis electrode 30F and the trans electrode 34F are at least substantially parallel to each other in an at least substantially horizontal direction. In other examples, the cis and trans electrodes can be in any suitable orientation relative to each other and to the nanopore device. The nanopore device 10F further includes a field effect transistor (FET) sensor 22F positioned between the first nanoscale opening 23F and the second nanoscale opening 25F. The FET sensor includes a source (S) 50F, a drain (D) 52F, and a channel 54F connecting the source 50F to the drain 52F. In some embodiments, the channel 54F has a nanowire configuration, similar to the structure shown in Figures 3B and 3C. In other embodiments, the channel 54F has a nanosheet configuration, similar to the structure shown in Figures 3D and 3E. The metal interconnects 66F and 64F are in electrical communication with the source 50F and the drain 52F of the FET 22F through the etch stop layer 38F. The metal interconnects communicate data from the FET sensor 22F to a control system that monitors the FET sensor 22F. In an alternative embodiment, the nanopore sequencing device 10F may use a porous structure in place of the second nanoscale opening 25F, similar to the structure illustrated in FIG.

[0115] As shown in the cross-sectional top view, FIGS. 7B and 7B', the fluid tunnel 21F is offset from the channel 54F. In other words, the fluid tunnel 21F does not extend through the channel 54F and is therefore not visible in the cross-sectional side view, FIG. 7A. Rather, the fluid tunnel 21F extends through the interlayer dielectric 68F around the channel 54F. In FIGS. 7B and 7B', the electrolyte 20F can be seen in the fluid tunnel 21F. The boundary of the fluid tunnel 21F can be circular as shown in FIGS. 7B and 7B'. In other embodiments, the boundary of the fluid tunnel 21F can be elliptical as shown in FIGS. 3D and 3E. Alternatively, the boundary of the fluid tunnel 21F can be of almost arbitrary shape and size. In some embodiments, the FET sensor 22F can include a stack of channels, similar to the structure illustrated in FIG. 6, but the fluid tunnel does not extend through the stack of channels.

[0116] One non-limiting advantage of the arrangement of offset fluid tunnels 21F with respect to channel 54F shown in Figures 7A, 7B, and 7B' is a simpler manufacturing process flow. Etching holes / openings in the channel may disturb the gate oxide of the device and require an additional oxide regrowth step. The embodiment shown in Figures 7A, 7B, and 7B' can avoid etching holes or openings in the source-drain channel.

[0117] The interlayer dielectric 68F can be any suitable insulator, such as SiO2, HfO2, or Al2O3. When the interlayer dielectric 68F is silicon dioxide, etching can be performed to etch various components of the nanopore sequencing device. For example, etching can be performed using an etchant with high anisotropy, such as a fluorinated reactive ion etchant, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar, as some non-limiting examples.

[0118] In one embodiment, the source, drain, and channel of FET sensor 22F may be formed of silicon, and the surface of the silicon may be thermally oxidized to form a gate oxide on the channel of FET sensor 22F.

[0119] In the nanopore sequencing device 10F shown in FIG. 7A, most of the material directly above the line 3-3 separating the channel 54F from the electrolyte 20F is removed, exposing the channel 54F of the FET sensor 22F to the electrolyte 20F. As shown in FIG. 7A, a portion of the channel 54F is exposed to the electrolyte from below. In other embodiments, similar to the structure shown in FIG. 4A, most of the material directly below the channel 54F can be removed or hollowed out, exposing a larger portion of the channel 54F to the electrolyte from below, which can be formed by undercutting the active area 54F of the FET sensor 22F by known methods. Only a thin layer of gate oxide 56F is grown around the channel 54F. The upper surface 55F and the lower surface 58F of the gate oxide 56F are fluidly exposed to the electrolyte 20F in the central well 15F. A thin layer of gate oxide 56F separates the channel 54F from the electrolyte 20F and exposes the channel 54F of the FET sensor 22F to the electrolyte 20F. The thickness of the gate oxide 56F can be from about 1 to about 10 nm, and in some embodiments, from about 2 to about 4 nm. The thickness of the gate oxide 56F is determined by the potential V M is selected such that a sufficiently strong electric field can be applied to induce an inversion layer of electrons or holes that form a conductive path at the channel 54F-gate oxide 56F boundary for conduction between the source 50F and the drain 52F.

[0120] The membrane 24F can be either a non-permeable or semi-permeable material. The first nanoscale opening 23F extends through the membrane 24F. It should be understood that the membrane 24F can be formed from any suitable natural or synthetic material, as described herein. In an embodiment, the membrane 24F is selected from the group consisting of lipids and biomimetic equivalents of lipids. In a further example, the membrane 24F is a synthetic membrane (e.g., a solid-state membrane, one example of which is silicon nitride), and the first nanoscale opening 23F is in a solid-state nanopore that extends through the membrane 24F. In an embodiment, the first nanoscale opening 23F extends through, for example, a polynucleotide nanopore, a polypeptide nanopore, or a solid-state nanopore, e.g., a carbon nanotube, disposed in the membrane.

[0121] The first nanopore 18F can be any of a biological nanopore, a solid-state nanopore, a hybrid nanopore, and a synthetic nanopore. In some embodiments, the first nanopore 18F has two open ends and a hollow core or hole (i.e., the first nanoscale opening 23F) connecting the two open ends. When inserted into the membrane 24F, one of the open ends of the first nanopore 18F faces the cis well 14F, and the other of the open ends of the first nanopore 18F faces the central well 15F. In some cases, the open end of the first nanopore 18F facing the central well 15F can be fluidly connected to the fluid tunnel 21F and aligned with at least a portion of the offset fluid tunnel 21F. In other examples, the open end of the first nanopore 18F facing the central well 15F is fluidly connected to the fluid tunnel 21F but is not aligned with the offset fluid tunnel 21F. The hollow core of the first nanopore 18F allows for fluidic and electrical connection between the cis well 14F and the central well 15F. The diameter of the hollow core of the first nanopore 18F can range from about 1 nm up to about 1 μm and can vary along the length of the first nanopore 18F. In some embodiments, the open end facing the cis well 14F can be larger than the open end facing the central well 15F. In other embodiments, the open end facing the cis well 14F can be smaller than the open end facing the central well 15F.

[0122] A method of using the nanopore sequencing device 10F may include introducing an electrolyte 20F into each of the cis well 14F, the trans well 16F, the central well 15F, and the fluid tunnel 21F. After introducing the electrolyte, the method may include providing a polynucleotide to be sequenced in the cis well 14F. After providing the polynucleotide, the method may include applying a voltage bias between the cis electrode 30F and the trans electrode 34F. The voltage bias drives the polynucleotide from the cis well 14F through the first nanoscale opening 23F to the central well 15F. As the polynucleotide passes through the first nanoscale opening 23F, the electrical resistance of the first nanoscale opening changes in response to the identity of the base in the polynucleotide at the first nanoscale opening. As a result, the potential (V) of the electrolyte 20F in the central well 15F (or equivalently, the offset fluid tunnel 21F) increases. M ) varies with the identity of the base. M ) is effectively the gate voltage applied to the FET, which adjusts the conductivity of channel 54F. Thus, measuring the response of the FET can determine the identity of the base.

[0123] Figure 8 illustrates yet another variation of a nanopore device in which the source-drain channels may not be etched to form fluidic tunnels, but instead utilize vertical field effect transistors, such that they are oriented vertically along the sides of the fluidic pathway through the device, as described below. Figure 8 is a cross-sectional side view of a vertical FET nanopore sequencing device 810G.

[0124] The nanopore sequencing device 810G shown in FIG. 8 includes a cis electrode 830G that connects to a cis well 814G. The cis well 814G has a bottom portion that includes a first nanopore 818G disposed in a membrane 824G. The first nanopore 818G includes a first nanoscale opening 823G defined by the first nanopore 818G in fluid communication with a second nanoscale opening 825G. The second nanoscale opening 825G can be disposed in a narrowed region 817G of a transwell 816G at the bottom portion of the device 810G. As shown, the second nanoscale opening is formed in a substrate material 862G. In other embodiments, the substrate material 862G does not have a narrowed region, similar to the structure shown in FIG. 3A, but is more planar in configuration. The first nanopore 818G provides a fluid path for the electrolyte 820G to pass between the ciswell 814G and the central well 815G. A substrate for sequencing may include an array of nanopore sequencing devices. In one embodiment of a nanopore sequencing device, the transwell is fluidically connected to the ciswell by the central well and the respective second and first nanoscale openings. In a substrate with an array of nanopore sequencing devices, one common ciswell and one common transwell may be in communication with some or all of the nanopore sequencing devices in the array on the substrate. However, it should be understood that an array of nanopore devices may also include several ciswells fluidly isolated from each other or fluidly isolated from each other and fluidly connected to a respective one or more transwells defined in the substrate. Multiple ciswells may be desired, for example, to allow for the measurement of multiple polynucleotides on a single substrate. In some embodiments, a substrate having an array of nanopore sequencing devices comprises one common cis electrode, one common trans electrode, one common cis well, one common trans well, and a plurality of nanopore sequencing devices such as that shown in FIG. 8, each nanopore sequencing device comprising a FET sensor and a dual pore having a first nanopore and a second nanopore.Each nanopore sequencing device of the multiple nanopore sequencing devices can measure the resistance or signal separately by its associated FET sensor. In other embodiments, each nanopore sequencing device can comprise multiple pores with three or more nanopores and FET sensors. In other embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis well, multiple trans wells, and multiple nanopore sequencing devices, each of which can be individually addressable with an individual trans electrode. In other embodiments, the substrate with the array of nanopore sequencing devices comprises multiple common cis wells, multiple trans wells, and multiple nanopore sequencing devices, each of which can be individually addressable with an individual trans electrode.

[0125] In one example, the cis electrode 830G and the trans electrode 834G are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis electrode and the trans electrode can be in any suitable orientation relative to each other and to the nanopore device. The nanopore device 810G further includes a vertical field effect transistor (FET) sensor positioned between the first nanoscale opening 823G and the second nanoscale opening 825G. The FET sensor includes a source (SRC) 850G, a drain (DRN) 852G, and a channel connecting the source to the drain. The FET channel is along a vertical direction, which is from the cis electrode 830G to the trans electrode 834G. In some embodiments, the channel has a nanowire configuration, similar to the structures shown in Figures 3B and 3C. In other embodiments, the channel has a nanosheet configuration, similar to the structures shown in Figures 3D and 3E. Metal interconnects 866G and 864G are in electrical communication with the source 850G and the drain 852G of the FET. The metal interconnects communicate data from the FET sensor to a control system that monitors the FET sensor. In an alternative embodiment, the nanopore sequencing device 810G may use a porous structure in place of the second nanoscale opening 825G, similar to the structure illustrated in FIG.

[0126] As shown in FIG. 8, the source 850G, channel, and drain 852G of the vertical FET sensor are stacked vertically. The vertical FET is arranged on a side of a central well 815g. In one embodiment, the source, drain, and channel of the FET sensor can be formed of silicon, and the surface of the silicon can be thermally oxidized to form a gate oxide 856G on the channel of the FET sensor. The vertical side of the gate oxide 856G is fluidly exposed to the electrolyte 820G in the central well 815G. The thin layer of gate oxide 856G separates the channel from the electrolyte 820G and exposes the channel of the FET sensor to the electrolyte 820G. The thickness of the gate oxide 856G can be about 1 to about 10 nm, and in some embodiments, about 2 to about 4 nm. The thickness of the gate oxide 856G is determined by the potential V Mis selected such that a sufficiently strong electric field can induce an inversion layer of electrons or holes that form a conductive path at the channel-gate oxide boundary to conduct between the source 850G and the drain 852G. In some embodiments, the FET sensor can include multiple vertical source-drain channels arranged in parallel along the sides of a central well.

[0127] One non-limiting advantage of a vertical FET sensor is that etching of a fluid tunnel through the FET channel may not be required. Etching holes / openings in the channel may disturb the gate oxide of the device and require an additional oxide regrowth step. An embodiment such as that shown in FIG. 8 with vertical FETs arranged on the side of the central well 815g can avoid etching holes or openings in the source-drain channel.

[0128] The interlayer dielectric 868G can be any suitable insulator, such as SiO2, HfO2, or Al2O3. When the interlayer dielectric 868G is silicon dioxide, etching can be performed to etch various components of the nanopore sequencing device. For example, etching can be performed using an etchant with high anisotropy, such as a fluorinated reactive ion etchant including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar, as some non-limiting examples.

[0129] The membrane 824G can be either a non-permeable or semi-permeable material. The first nanoscale opening 823G extends through the membrane 824G. It should be understood that the membrane 824G can be formed from any suitable natural or synthetic material, as described herein. In an embodiment, the membrane 824G is selected from the group consisting of lipids and biomimetic equivalents of lipids. In a further example, the membrane 824G is a synthetic membrane (e.g., a solid-state membrane, one example of which is silicon nitride), and the first nanoscale opening 823G is in a solid-state nanopore that extends through the membrane 824G. In an embodiment, the first nanoscale opening 823G extends through, for example, a polynucleotide nanopore, a polypeptide nanopore, or a solid-state nanopore, e.g., a carbon nanotube, disposed in the membrane.

[0130] The first nanopore 818G can be any of a biological nanopore, a solid-state nanopore, a hybrid nanopore, and a synthetic nanopore. In some embodiments, the first nanopore 818G has two open ends and a hollow core or hole (i.e., the first nanoscale opening 823G) that connects the two open ends. When inserted into the membrane 824G, one of the open ends of the first nanopore 818G faces the cis well 814G and the other of the open ends of the first nanopore 818G faces the central well 815G. The hollow core of the first nanopore 818G allows for fluidic and electrical connection between the cis well 814G and the central well 815G. The diameter of the hollow core of the first nanopore 818G can range from about 1 nm up to about 1 μm and can vary along the length of the first nanopore 818G. In some embodiments, the open end facing cis-well 814G can be larger than the open end facing central well 815G. In other embodiments, the open end facing cis-well 814G can be smaller than the open end facing central well 815G.

[0131] A method of using the nanopore sequencing device 810G may include introducing an electrolyte 820G into each of the cis well 814g, the trans well 816G, and the central well 815g. After introducing the electrolyte, the method may include providing a polynucleotide to be sequenced in the cis well 814G. After providing the polynucleotide, the method may include applying a voltage bias between the cis electrode 830G and the trans electrode 834G. The voltage bias drives the polynucleotide from the cis well 814G through the first nanoscale opening 823G to the central well 815G. As the polynucleotide passes through the first nanoscale opening 823G, the electrical resistance of the first nanoscale opening changes in response to the identity of the base in the polynucleotide at the first nanoscale opening. As a result, the potential (V M ) varies with the identity of the base. M ) is effectively the gate voltage applied to the FET, which modulates the conductivity of the FET channel. Thus, measuring the response of the FET can determine the identity of the base.

[0132] FIG. 9 illustrates yet another further variation of a nanopore sequencing device having a field effect transistor (FET) with a non-Faradic metal electrode. In this embodiment, the FET has a non-Faradic metal electrode with a metal structure that does not participate in the faradaic process in the nanopore sequencing device, i.e., the electrochemical reaction does not occur at the metal structure. The non-Faradic metal electrode is used to detect the potential of the electrolyte in the central well and transmit the potential to the FET as a detected signal. This design means that the FET can detect the potential of the electrolyte but is not exposed to the electrolyte. FIG. 9 is a cross-sectional side view of a nanopore sequencing device 910H.

[0133] The nanopore sequencing device 910H shown in FIG. 9 includes a cis electrode 930H that connects to a cis well 914H. The cis well 914H has a bottom portion that includes a first nanopore 918H disposed in a membrane 924H. The first nanopore 918H includes a first nanoscale opening 923H defined by the first nanopore 918H in fluid communication with a second nanoscale opening 925H. The second nanoscale opening 925H can be disposed in a narrowed region 917H of a transwell 916H at the bottom portion of the device 910H. As shown, the second nanoscale opening is formed in a substrate material 962H. In other embodiments, the substrate material 962H does not have a narrowed region, similar to the structure shown in FIG. 3A, but is more planar in configuration. A first nanopore 918H provides a fluid pathway for electrolyte 920H to pass between cis well 914H and central well 915H.

[0134] In one example, the cis electrode 930H and the trans electrode 934H are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis and trans electrodes can be in any suitable orientation relative to each other and to the nanopore device. The nanopore device 910H further includes a field effect transistor (FET) sensor 922H positioned between the first nanoscale opening 923H and the second nanoscale opening 925H. The FET sensor 922H includes a source (SRC) 950H, a drain (DRN) 952H, and a channel 954H connecting the source 950H to the drain 952H. The FET channel can be along a horizontal direction. In some embodiments, the FET channel has a nanowire configuration, similar to the structure shown in Figures 3B and 3C. In other embodiments, the FET channel has a nanosheet configuration, similar to the structure shown in Figures 3D and 3E. Metal interconnects 966H and 964H are in electrical communication with the source 950H and drain 952H of the FET. Metal interconnects 966H and 964H communicate data from the FET sensor to a control system (presently shown) that monitors the FET sensor. In an alternative embodiment, the nanopore sequencing device 910H may use a porous structure in place of the second nanoscale opening 925H, similar to the structure illustrated in FIG. 5A.

[0135] As shown in FIG. 9, the FET sensor 922H is not in direct contact with the electrolyte. In one embodiment, the source, drain, and channel of the FET sensor may be formed of silicon, and the surface of the silicon may be thermally oxidized to form a gate oxide 956H on the channel of the FET sensor. As shown in FIG. 9, the gate oxide 956H is not fluidly exposed to the electrolyte 920H in the central well 915H. Instead, a non-Faraday metal electrode structure 999H is exposed to the electrolyte. The metal structure 999H is in direct contact with the electrolyte potential V of the electrolyte in the central well. Mand transmits the detected signal to the FET. Relative to the size of the central well, which is on the order of a few microns, the path length or characteristic size of the metal structure 999H can be about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, or any value therebetween.

[0136] The size and shape of the metal structure 999H may be appropriately selected to avoid high parasitic capacitance in the system. The metal structure 999H may be made of a non-Faraday corrosion resistant metal with respect to the electrolyte. The metal structure 999H may be made of platinum, iridium, ruthenium, palladium, tantalum, gold, or any combination thereof. Electrochemical reactions may not occur in the metal structure. In some embodiments, the metal structure 999H may have a cup-shaped portion exposed to the electrolyte to increase the contact area with the electrolyte. In some embodiments, the portion of the metal structure 999H exposed to the electrolyte may include one or more holes or openings. In some embodiments, the portion of the metal structure 999H exposed to the electrolyte may include several parallel fins to increase the contact area with the electrolyte, and the fins may be partially vertically or horizontally arranged. Using the metal structure 999H to contact the electrolyte allows for the separation of the FET from the central well, which may be easier to manufacture in some embodiments. Separating the size of the FET with the size of the central well also allows for larger FETs, which may allow for higher signal detection sensitivity and lower noise levels. This configuration also allows for decoupling the size of the FET, which determines the limit of signal detection, from the size of the metal structure 999H, thus providing more design flexibility.

[0137] The thickness of the gate oxide 956H can be from about 1 to about 10 nm, and in some embodiments, from about 2 to about 4 nm. The thickness of the gate oxide 956H is determined by the potential V M The dielectric constant is selected such that a sufficiently strong electric field can induce an inversion layer of electrons or holes that form a conductive path at the channel-gate oxide boundary to conduct between the source 950H and the drain 952H. The interlayer dielectric 968H can be any suitable insulator, such as SiO2, HfO2, or Al2O3. When the interlayer dielectric 968H is silicon dioxide, etching can be performed to etch various components of the nanopore sequencing device. For example, etching can be performed using an etchant with high anisotropy, such as a fluorinated reactive ion etch, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar, as some non-limiting examples.

[0138] The membrane 924H can be either a non-permeable or semi-permeable material. The first nanoscale opening 923H extends through the membrane 924H. It should be understood that the membrane 924H can be formed from any suitable natural or synthetic material, as described herein. In an embodiment, the membrane 924H is selected from the group consisting of lipids and biomimetic equivalents of lipids. In a further example, the membrane 924H is a synthetic membrane (e.g., a solid-state membrane, one example of which is silicon nitride), and the first nanoscale opening 923H is in a solid-state nanopore that extends through the membrane 924H. In an embodiment, the first nanoscale opening 923H extends through, for example, a polynucleotide nanopore, a polypeptide nanopore, or a solid-state nanopore, e.g., a carbon nanotube, disposed in the membrane. The first nanopore 918H can be any of a biological nanopore, a solid-state nanopore, a hybrid nanopore, and a synthetic nanopore. In some embodiments, the first nanopore 918H has two open ends and a hollow core or hole (i.e., the first nanoscale opening 923H) connecting the two open ends. When inserted into the membrane 924H, one of the open ends of the first nanopore 918H faces the cis well 914H and the other of the open ends of the first nanopore 918H faces the central well 915H. The hollow core of the first nanopore 918H allows for fluidic and electrical connection between the cis well 914H and the central well 915H. The diameter of the hollow core of the first nanopore 918H can range from about 1 nm up to about 1 μm and can vary along the length of the first nanopore 918H. In some embodiments, the open end facing the cis well 914H can be larger than the open end facing the central well 915H. In other embodiments, the open end facing the central well 914H can be smaller than the open end facing the central well 915H.

[0139] A method of using the nanopore sequencing device 910H can include introducing an electrolyte 920H into each of the cis well 914H, the trans well 916H, and the central well 915H. After introducing the electrolyte, the method can include providing a polynucleotide to be sequenced into the cis well 914H. After providing the polynucleotide, the method can include applying a voltage bias between the cis electrode 930H and the trans electrode 934H. In some embodiments, the voltage bias can drive the polynucleotide from the cis well 914H through the first nanoscale opening 923H to the central well 915H. As the polynucleotide passes through the first nanoscale opening 923H, the electrical resistance of the first nanoscale opening changes in response to the identity of the base in the polynucleotide at the first nanoscale opening. In an alternative embodiment, the polynucleotide does not pass through the first nanoscale opening, but a nucleotide tag or label incorporated by a polymerase acting on the polynucleotide may pass through the first nanoscale opening or may be temporarily present in the first nanoscale opening. Thus, the electrical resistance of the first nanoscale opening changes in response to the identity of the incorporated nucleotide, which is complementary to the identity of the base in the polynucleotide. As a result, the potential (V M ) varies with the identity of the base in the polynucleotide. M ) is effectively the gate voltage applied to the FET, which modulates the conductivity of the FET channel. Thus, measuring the response of the FET can determine the identity of the bases in a polynucleotide.

[0140] A substrate for sequencing may include an array of nanopore sequencing devices, such as the one shown in FIG. 9. In one embodiment of a nanopore sequencing device, the transwell is fluidically connected to the ciswell by a central well and respective second and first nanoscale openings. In a substrate with an array of nanopore sequencing devices, one common ciswell and one common transwell may be in communication with some or all of the nanopore sequencing devices in the array on the substrate. However, it should be understood that an array of nanopore devices may also include several ciswells that are fluidically isolated from each other, or fluidically isolated from each other and fluidically connected to a respective one or more transwells defined in the substrate. Multiple ciswells may be desired, for example, to allow for the measurement of multiple polynucleotides on a single substrate. In some embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis electrode, one common trans electrode, one common cis well, one common trans well, and a plurality of nanopore sequencing devices such as those shown in FIG. 9, where each nanopore sequencing device comprises a dual pore with a FET sensor and a first nanopore and a second nanopore. Each nanopore sequencing device of the plurality of nanopore sequencing devices can measure resistance or signal separately by its associated FET sensor. In other embodiments, each nanopore sequencing device can comprise a plurality of pores with three or more nanopores and FET sensors. In other embodiments, the substrate with the array of nanopore sequencing devices comprises one common cis well, a plurality of trans wells, and a plurality of nanopore sequencing devices, where each nanopore sequencing device can be individually addressable with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of common cis wells, a plurality of trans wells, and a plurality of nanopore sequencing devices, each of which may be individually addressable with an individual trans electrode.

[0141] definition All technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless defined otherwise.

[0142] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a sequence" can include a plurality of such sequences, and so forth.

[0143] The terms comprising, including, and containing, and various forms of these terms, are intended to be synonymous with each other and are equally broad. Furthermore, unless expressly stated to the contrary, an embodiment that "comprising," "including," or "having" an element or elements having a particular characteristic may include the additional elements, regardless of whether the additional elements have that characteristic.

[0144] As used herein, terms such as "fluidly connecting," "fluid communication," "fluidically coupled," and the like refer to two spatial regions that are connected together such that liquid or gas can flow between the two spatial regions. For example, a syswell may be fluidly connected to a transwell by a central well, a fluid tunnel, and a narrowed region such that at least a portion of the electrolyte can flow between the connected wells. The two spatial regions may be in fluid communication through first and second nanoscale openings, or through one or more valves, restrictions, or other fluidic components to control or regulate the flow of fluid through the system.

[0145] As used herein, the term "interstitial region" refers to an area in a substrate / solid support or membrane, or an area on a surface that separates other areas, regions, features associated with the support or membrane or surface. For example, an interstitial region of a membrane can separate one nanopore of an array from another nanopore of the array. For another example, an interstitial region of a substrate can separate one transwell from another transwell. Two areas separated from each other can be distinct, i.e., lacking physical contact with each other. In many examples, the interstitial region is continuous, whereas the areas are distinct, such as in the case of multiple nanopores defined in an otherwise continuous membrane, or multiple wells defined in an otherwise continuous substrate / support. The separation provided by the interstitial region can be partial or complete. The interstitial region may have a surface material that is different from the surface material of the features defined in the surface. For example, the surface material in the interstitial region can be a lipid material, and the nanopores formed in the lipid material can have an amount or concentration of polypeptide that exceeds the amount or concentration present in the interstitial region. In some examples, the polypeptide may not be present in interstitial regions.

[0146] As used herein, the term "membrane" refers to an impermeable or semi-permeable barrier or other sheet that separates two liquid / gel chambers (e.g., a syswell and a fluid cavity) that may contain the same or different compositions therein. The permeability of the membrane to any given species depends on the properties of the membrane. In some examples, the membrane may be impermeable to ions, to electrical current, and / or to fluids. For example, a lipid membrane may be impermeable to ions (i.e., does not allow any ion transport therethrough) but may be at least partially permeable to water (e.g., water diffusivity ranges from about 40 μm / s to about 100 μm / s). For another example, a synthetic / solid-state membrane, one example of which is silicon nitride, may be impermeable to ions, charges, and fluids (i.e., all diffusion of these species is zero). Any membrane may be used in accordance with the present disclosure as long as the membrane can include nanoscale openings through the membrane and can maintain a potential difference across the membrane. The membrane may be a single layer or a multi-layer membrane. A multi-layer membrane includes two or more layers, each of which is a non-permeable or semi-permeable material.

[0147] Membranes can be formed of materials of biological or non-biological origin. Biologically derived materials refer to materials derived or isolated from biological environments, such as organisms or cells, or synthetically produced versions of biologically available structures (e.g., biomimetic materials).

[0148] Exemplary membranes made from biologically derived materials include monolayers formed by bolalipids. Another exemplary membrane made from biologically derived materials includes lipid bilayers. Suitable lipid bilayers include, for example, cell membranes, organelle membranes, liposomes, planar lipid bilayers, and supported lipid bilayers. Lipid bilayers can be formed, for example, from two opposing layers of phospholipids arranged such that the hydrophilic head groups of the lipids face outward toward the aqueous environment on each side of the bilayer, whereas the hydrophobic tail groups of the lipids face toward each other to form a hydrophobic interior. Lipid bilayers can also be formed, for example, by a method of carrying the lipid monolayer at an aqueous / air interface over either side of an aperture substantially perpendicular to the interface. The lipid is usually added to the surface of an aqueous electrolyte solution by first dissolving it in an organic solvent and then allowing a small amount of solvent to evaporate on the surface of the aqueous solution on both sides of the aperture. Once the organic solvent has at least partially evaporated, the solution / air interface on either side of the aperture is physically moved up and down across the aperture until a bilayer is formed. Other suitable methods of bilayer formation include tip-dipping of liposomal bilayers, painting bilayers, and patch-clamping. Any other method for obtaining or generating lipid bilayers may also be used.

[0149] Non-biological materials can also be used as membranes. Some of these materials are solid-state materials and can form solid-state membranes, while others of these materials can form thin liquid films or membranes. Solid-state membranes can be single layers, such as coatings or films on a supporting substrate (i.e., solid support), or free-standing elements. Solid-state membranes can also be composites of multi-layer materials in a sandwich configuration. Any non-biological material can be used as long as the resulting membrane can contain nanoscale openings through the membrane and can maintain a potential difference across the membrane. The membrane can include organic materials, inorganic materials, or both. Examples of suitable solid-state materials include, for example, microelectronic materials, insulating materials (e.g., silicon nitride (Si3N4), aluminum oxide (Al2O3), hafnium oxide (HfO2), tantalum pentoxide (Ta2O5), silicon oxide (SiO2), etc.), some organic and inorganic polymers (e.g., plastics such as polyamides, polytetrafluoroethylene (PTFE), or elastomers such as two-component addition-cured silicon rubber), and glass. In addition, the solid-state membrane can be made from a monolayer of graphene, which is an atomically thin sheet of carbon atoms densely packed in a two-dimensional honeycomb lattice, a multilayer of graphene, or one or more layers of graphene mixed with one or more layers of other solid-state materials. The graphene-containing solid-state membrane can include at least one graphene layer that is a graphene nanoribbon or a graphene nanogap, which can be used as an electrical sensor to characterize the target polynucleotide. It should be understood that the solid-state membrane can be made by any suitable method, for example, chemical vapor deposition (CVD). In an embodiment, the graphene membrane can be prepared by either CVD or exfoliation from graphite. Examples of suitable thin liquid film materials that can be used include diblock or triblock copolymers, for example, amphiphilic PMOXA-PDMS-PMOXA ABA triblock copolymers.

[0150] As used herein, the term "nanopore" is intended to mean a hollow structure extending from or across a membrane that allows ions, current, and / or fluids to pass from one side of the membrane to the other side of the membrane. For example, a membrane that inhibits the passage of ions or water-soluble molecules can include a nanopore structure extending across the membrane to allow the passage of ions or water-soluble molecules from one side of the membrane to the other side of the membrane (through a nanoscale opening extending through the nanopore structure). The diameter of the nanoscale opening extending through the nanopore structure can vary along its length (i.e., from one side of the membrane to the other side of the membrane), but is at any point in the nanoscale (i.e., from about 1 nm to about 100 nm, or less than 1000 nm). Examples of nanopores include, for example, biological nanopores, solid-state nanopores, and biological and solid-state hybrid nanopores.

[0151] As used herein, the term "diameter" is intended to mean the longest straight line isolatable in the cross-section of a nanoscale opening that passes through the geometric center of the cross-section of the nanoscale opening. It is understood that the nanoscale opening may or may not have a circular or substantially circular cross-section (the cross-section of the nanoscale opening that is substantially parallel to the cis / trans electrodes). Furthermore, the cross-section may be of a regular or irregular shape.

[0152] As used herein, the term "biological nanopore" is intended to mean a nanopore whose structural portions are made from materials of biological origin. Biological origin refers to materials derived or isolated from a biological environment, such as an organism or cell, or synthetically produced versions of biologically available structures. Biological nanopores include, for example, polypeptide nanopores and polynucleotide nanopores.

[0153] As used herein, the term "polypeptide nanopore" is intended to mean a protein / polypeptide that extends across a membrane and allows ions, electrical current, polymers such as DNA or peptides, or other molecules of appropriate size and charge, and / or fluids to flow from one side of the membrane to the other side of the membrane. Polypeptide nanopores can be monomeric, homopolymeric, or heteropolymeric. Polypeptide nanopore structures include, for example, α-helix bundle nanopores and β-barrel nanopores. Examples of polypeptide nanopores include α-hemolysin, Mycobacterium smegmatis porin A (MspA), gramidiin A, maltoporin, OmpF, OmpC, PhoE, Tsx, F pilus, and the like. The protein α-hemolysin is naturally found in cell membranes and functions as a pore for ions or molecules to be transported in and out of the cell. Mycobacterium smigmatis porin A (MspA) is a membrane porin produced by mycobacteria that allows hydrophilic molecules to enter the bacteria. MspA forms a tightly interconnected octamer and transmembrane beta-barrel that resembles a goblet and contains a central pore.

[0154] A polypeptide nanopore can be synthetic. A synthetic polypeptide nanopore comprises a protein-like amino acid sequence that does not occur in nature. A protein-like amino acid sequence can include some of the amino acids that are known to exist but do not form the basis of a protein (i.e., non-proteinogenic amino acids). A protein-like amino acid sequence can be artificially synthesized rather than expressed in an organism and then purified / isolated.

[0155] As used herein, the term "polynucleotide nanopore" is intended to include a polynucleotide that extends across a membrane and allows ions, current, and / or fluids to flow from one side of the membrane to the other side of the membrane. A polynucleotide pore can include, for example, polynucleotide origami (e.g., the nanoscale folding of DNA to create a nanopore).

[0156] Also, as used herein, the term "solid-state nanopore" is intended to mean a nanopore whose structural portion is defined by a solid-state membrane and comprises non-biological (i.e., non-biological) material. Solid-state nanopores can be formed of inorganic or organic materials. Solid-state nanopores include, for example, silicon nitride nanopores, silicon dioxide nanopores, and graphene nanopores.

[0157] The nanopores disclosed herein may be hybrid nanopores. "Hybrid nanopore" refers to a nanopore that contains materials of both biological and non-biological origin. Examples of hybrid nanopores include polypeptide solid-state hybrid nanopores and polynucleotide solid-state nanopores.

[0158] As used herein, the term "nanopore sequencer" refers to any of the devices disclosed herein that can be used for nanopore sequencing. In the embodiments disclosed herein, during nanopore sequencing, the nanopore is immersed in an embodiment of an electrolyte disclosed herein, and a potential difference is applied across the membrane. In embodiments, the potential difference is an electrical potential difference or an electrochemical potential difference. The electrical potential difference can be imposed across the membrane via a voltage source that injects or applies a current to at least one of the ions of the electrolyte contained in one or more of the cis well or trans well. The electrochemical potential difference can be established by a difference in the ionic composition of the cis and trans wells combined with the electrical potential. The different ionic composition can be, for example, different ions in each well or different concentrations of the same ions in each well.

[0159] Application of a potential difference across the nanopore can force translocation of the nucleic acid through a first nanoscale opening 23 (e.g., shown in FIG. 2A and described in more detail below). One or more signals are generated corresponding to the translocation of the nucleotide through the nanopore. Thus, upon passing a target polynucleotide, or a mononucleotide or a probe derived from a target polynucleotide or mononucleotide, through the nanopore, the current across the membrane changes, for example, due to a base-dependent (or probe-dependent) blockage of the constriction. The signal from that change in current can be measured using any of a variety of methods. Each signal is unique to the species of nucleotide (or probe) in the nanopore, such that the resulting signal can be used to determine a characteristic of the polynucleotide. For example, the identity of one or more species of nucleotide (or probe) that generate a characteristic signal can be determined.

[0160] As used herein, a "nucleotide" comprises a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. A nucleotide is a monomeric unit of a nucleic acid sequence. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (RNA), the sugar is ribose, and in deoxyribonucleotides (DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present at the 2' position of the ribose. The nitrogen-containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of the deoxyribose is attached to the N-1 of the pyrimidine or the N-9 of the purine. The phosphate groups can be in mono-, di-, or triphosphate form. These nucleotides are naturally occurring nucleotides, however, it is further understood that non-naturally occurring nucleotides, modified nucleotides, or analogs of the aforementioned nucleotides can also be used.

[0161] As used herein, the term "signal" is intended to mean an indication that represents information. Signals include, for example, electrical signals and optical signals. The term "electrical signal" refers to an indication of an electrical quality that represents information. The indication can be, for example, current, voltage, tunneling, resistance, potential, voltage, conductance, or lateral electrical effect. "Electronic current" or "electric current" refers to the flow of charge. In an embodiment, the electrical signal can be a current passing through a nanopore, and a current can flow when a current difference is applied across the nanopore.

[0162] The term "substrate" refers to a rigid solid support that is insoluble in aqueous liquids and cannot pass liquids that are not present in apertures, ports, or other similar liquid conduits. In the embodiments disclosed herein, the substrate may have wells or chambers defined therein. Examples of suitable substrates include glass and modified or functionalized glass, plastics (acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (PTFE) (such as TEFLON® from Chemours), cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon, ceramic, silica or silica-based materials, silicon and modified silicon, carbon, metals, inorganic glasses, and fiber optic bundles.

[0163] Terms such as top, bottom, lower, upper, on, etc. are used herein to describe the device / nanopore sequencer and / or various components of the device. It is understood that these directional terms are not meant to indicate a particular orientation, but are used to specify the relative orientation between components. The use of directional terms is not to be construed as limiting the examples disclosed herein to any particular orientation. As used herein, terms such as "top", "bottom", "vertical", "horizontal", etc. are meant to indicate a relative orientation.

[0164] As used herein, the terms "well", "cavity" and "chamber" are used interchangeably and refer to discrete features defined in a device that can contain fluid (e.g., liquid, gel, gas). A cis well is a chamber that contains or is partially defined by a cis electrode, which in turn is fluidically connected to the fluidic system of the FET, which is fluidically connected to the transwell / chamber. An example of an array of the device can have one cis well or multiple cis wells. A trans well is a single chamber that contains or is partially defined by its own trans electrode, and is also fluidically connected to a cis well. In examples that include multiple trans wells, each trans well is electrically isolated from each other trans well. It should further be understood that a cross section of a well taken parallel to the surface of the substrate that at least partially defines the well can be curved, square, polygonal, hyperbolic, conical, angular, etc.

[0165] As used herein, a "field effect transistor" or "FET" is typically formed of a semiconductor material, e.g., silicon, germanium, gallium arsenide, silicon carbide, etc., and includes doped source / drain regions separated by a channel region. An n-FET is a FET with an n-channel where the current carriers are electrons. A p-FET is a FET with a p-channel where the current carriers are holes. The source / drain regions of an n-FET device may include a different material than the source / drain regions of a p-FET device. In some embodiments, the source / drain regions or the channel may be undoped. A doped region may be formed by adding dopant atoms to an intrinsic semiconductor. This changes the electron and hole carrier concentration of the intrinsic semiconductor at thermal equilibrium. A doped region may be p-type or n-type. As used herein, "p-type" refers to the addition of an impurity to an intrinsic semiconductor that creates a deficiency of valence electrons. For silicon, exemplary p-type dopants, i.e., impurities, include, but are not limited to, boron, aluminum, gallium, and indium. As used herein, "n-type" refers to the addition of an impurity that contributes free electrons to an intrinsic semiconductor. For silicon, exemplary n-type dopants, or impurities, include, but are not limited to, antimony, arsenic, and phosphorus. The dopants may be introduced by ion implantation or plasma doping.

[0166] For example, in an integrated circuit having a number of metal oxide semiconductor field effect transistors (MOSFETs), each MOSFET has a source and a drain formed in an active region of the semiconductor layer by implanting n-type or p-type impurities into the layer of semiconductor material. Disposed between the source and drain is a channel (or body) region. Disposed above the body region is a gate electrode. The gate electrode and the body are separated by a gate dielectric (gate oxide) layer. The channel region connects the source and drain, and current flows through the channel region from the source to the drain. Current flow is induced in the channel region by a voltage applied to the gate electrode.

[0167] Non-planar transistor device architectures such as nanosheet (or nanowire) transistors can provide increased device density and increased performance over planar transistors. A "gate all around" transistor is one in which the gate is configured to wrap around the channel. A "nanosheet transistor" refers to a type of FET that can include multiple stacked nanosheets that extend between a pair of source / drain regions and form a channel. In contrast to traditional planar FETs, nanosheet transistors can include a gate stack that wraps around the entire periphery of multiple nanosheet channel regions. The nanosheet transistor configuration allows for complete depletion in the nanosheet channel region, reducing shorting effects. A "nanowire transistor" can be similar to a nanosheet transistor, except that the channel can include nanowires instead of nanosheets. The all around gate structure in nanosheet or nanowire transistors can provide very small devices with better switching control, lower leakage current, faster operation, and lower output resistance.

[0168] A method to increase channel conductivity and reduce FET size is to form the channel as a nanostructure. For example, the gate-all-around (GAA) nanosheet FET is an architecture to provide a relatively small FET footprint by forming the channel region as a series of nanosheets. In the GAA configuration, a nanosheet-based FET includes a source region, a drain region, and a stacked nanosheet channel between the source and drain regions. A gate surrounds the stacked nanosheet channel and regulates the flow of electrons through the nanosheet channel between the source and drain regions. A GAA nanosheet FET can be fabricated by forming alternating layers of channel nanosheets and sacrificial nanosheets. The sacrificial nanosheets are released from the channel nanosheets before the FET device is completed. For n-type FETs, the channel nanosheets are typically silicon (Si) and the sacrificial nanosheets are typically silicon germanium (SiGe). For p-type FETs, the channel nanosheets are typically SiGe and the sacrificial nanosheets are typically Si. In some implementations, the channel nanosheet of the p-FET can be SiGe or Si, and the sacrificial nanosheet can be Si or SiGe. Forming the GAA nanosheet from alternating layers of channel nanosheets formed from a first type of semiconductor material (e.g., Si for n-type FETs and SiGe for p-type FETs) and sacrificial nanosheets formed from a second type of semiconductor material (e.g., SiGe for n-type FETs and Si for p-type FETs) provides excellent channel electrostatic control that is beneficial for continuously scaling gate lengths to 7 nanometer CMOS technology and below. The use of multilayer SiGe / Si sacrificial / channel nanosheets (or Si / SiGe sacrificial / channel nanosheets) to form the channel region in GAAFET semiconductor devices provides desirable device characteristics, including the introduction of strain at the interface between SiGe and Si.

[0169] In some embodiments, a "nanowire" is characterized by a critical dimension less than about 30 nm, while a "nanosheet" is characterized by a critical dimension greater than or equal to about 30 nm. In the exemplary device, the critical dimension is measured along the gate. In that direction, when the channel width is small, the channel cross-section is "wire"-like, whereas when the channel width is large, the channel cross-section is "sheet"-like.

[0170] In some embodiments, the smallest dimension of the nanosheets or nanowires is about 1-10, about 1-50, about 1-100, about 1-500, or about 1-1000 nm. In some embodiments, the smallest dimension of the nanosheets or nanowires is about 1-5, about 3-10, about 5-15, about 10-20, about 15-30, about 20-40, about 30-50, about 40-75, about 50-100, about 75-150, about 100-200, about 150-300, about 200-400, about 300-500, about 400-750, or about 500-1000 nm. In some embodiments, the smallest dimension of a nanosheet is at least about 3, about 5, about 7, about 10, about 15, about 20, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 2500, about 3000, about 4000, or about 5000 times smaller than the other two dimensions of the nanosheet. In some embodiments, the smallest dimension of the nanosheet is about 2-5, about 3-7, about 5-10, about 7-15, about 10-20, about 15-50, about 20-100, about 50-150, about 100-200, about 150-250, about 200-300, about 250-350, about 300-400, about 350-450, about 400-500, about 450-600, about 500-700, about 600-800, about 700-900, about 800-1000, about 900-2000, about 1000-2500, about 2000-3000, about 2500-4000, or about 3000-5000 times smaller than the other two dimensions of the nanosheet. In some embodiments, the smallest dimension of a nanosheet is at most about 3, about 5, about 7, about 10, about 15, about 20, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 2500, about 3000, about 4000, or about 5000 times smaller than the other two dimensions of the nanosheet.In some embodiments, the largest dimension of the nanowire is at least about 3, about 5, about 7, about 10, about 15, about 20, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 2500, about 3000, about 4000, or about 5000 times greater than the other two dimensions of the nanowire. In some embodiments, the maximum dimension of the nanowire is about 2-5, about 3-7, about 5-10, about 7-15, about 10-20, about 15-50, about 20-100, about 50-150, about 100-200, about 150-250, about 200-300, about 250-350, about 300-400, about 350-450, about 400-500, about 450-600, about 500-700, about 600-800, about 700-900, about 800-1000, about 900-2000, about 1000-2500, about 2000-3000, about 2500-4000, or about 3000-5000 times greater than the other two dimensions of the nanowire. In some embodiments, the largest dimension of the nanowire is at most about 3, about 5, about 7, about 10, about 15, about 20, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 2500, about 3000, about 4000, or about 5000 times greater than the other two dimensions of the nanowire.

[0171] As used herein, a "porous structure" or "frit" refers to a body having a pore portion. Typical pore diameters of the pore portion are, for example, about 100 μm or less, about 50 μm or less, about 10 μm or less, about 5 μm or less, about 1 μm or less, about 500 nm or less, about 100 nm or less, about 50 nm or less, about 10 nm or less, about 5 nm or less, about 1 nm or less, about 500 Å or less, about 100 Å or less, about 50 Å or less, about 10 Å or less, about 5 Å or less, about 10 Å or less, about 5 Å or less, about 100 μm or more, about 5 μm or more, about 1 μm or more, about 500 nm or more, about 100 nm or more, about 50 nm or more, about 10 nm or more, about 5 nm or more, about 1 nm or more, about 500 Å or more, about 100 Å or more, about 50 Å or more. or more, about 10 Å or more, about 5 Å or more, about 500 to about 100 μm, about 250 to about 50 μm, about 125 to about 25 μm, about 50 to about 10 μm, about 25 to about 5 μm, about 12.5 to about 2.5 μm, about 5.5 to about 0.5 μm, about 500 to about 100 nm, about 250 to about 50 nm, about 125 to about 25 nm, about 50 to about 10 nm, about 25 to about 5 nm, about 12.5 to about 2.5 nm, about 5.5 to about 0.5 nm, about 500 to about 100 Å, about 250 to about 50 Å, about 125 to about 25 Å, about 50 to about 10 Å, about 25 to about 5 Å, about 12.5 to about 2.5 Å, or about 5.5 to about 1 Å. There may be a distribution of different pore diameters.

[0172] The porous structure may be formed of a porous material comprising a matrix that defines an array of pores with sufficient porosity to allow the desired function of the porous material. As used herein, the term "porosity" refers to the amount of voids in a porous material comprising a matrix. Thus, the total volume of a porous material comprising a matrix is ​​based on the matrix space and voids. As used herein, the term "voids" refers to the actual or physical space in a porous material comprising a matrix. Thus, the total volume of a porous material comprising a matrix disclosed herein is based on the matrix space and voids.For example, a porous material comprising a matrix defining an array of pores may have a volume of at least about 40% of the total volume of the matrix, at least about 50% of the total volume of the matrix, at least about 60% of the total volume of the matrix, at least about 70% of the total volume of the matrix, at least about 80% of the total volume of the matrix, at least about 90% of the total volume of the matrix, at least about 95% of the total volume of the matrix, or at least about 97% of the total volume of the matrix, at most about 40% of the total volume of the matrix, at most about 50% of the total volume of the matrix, at most about 60% of the total volume of the matrix, at most about 70% of the total volume of the matrix, at most ... 0%, at most about 90% of the total volume of the matrix, at most about 95% of the total volume of the matrix, or at most about 97% of the total volume of the matrix, about 40% to about 97% of the total volume of the matrix, about 50% to about 97% of the total volume of the matrix, about 60% to about 97% of the total volume of the matrix, about 70% to about 97% of the total volume of the matrix, about 80% to about 97% of the total volume of the matrix, about 90% to about 97% of the total volume of the matrix, about 40% to about 95% of the total volume of the matrix, The porosity may be about 50% to about 95% of the total volume of the matrix, about 60% to about 95% of the total volume of the matrix, about 70% to about 95% of the total volume of the matrix, about 80% to about 95% of the total volume of the matrix, about 90% to about 95% of the total volume of the matrix, about 40% to about 90% of the total volume of the matrix, about 50% to about 90% of the total volume of the matrix, about 60% to about 90% of the total volume of the matrix, about 70% to about 90% of the total volume of the matrix, or about 80% to about 90% of the total volume of the matrix.For example, a porous material comprising a matrix defining an array of pores may be, for example, about 50% of the total volume of the matrix, about 60% of the total volume of the matrix, about 70% of the total volume of the matrix, about 80% of the total volume of the matrix, about 90% of the total volume of the matrix, about 95% of the total volume of the matrix, or about 97% of the total volume of the matrix, at least about 50% of the total volume of the matrix, at least about 60% of the total volume of the matrix, at least about 70% of the total volume of the matrix, at least about 80% of the total volume of the matrix, at least about 90% of the total volume of the matrix, at least about 95% of the total volume of the matrix, or at least about 97% of the total volume of the matrix, at most about 50% of the total volume of the matrix, at most about 60% of the total volume of the matrix, at most about 70% of the total volume of the matrix, at most about The matrix may have voids of at most about 80%, at most about 90% of the total matrix volume, at most about 95% of the total matrix volume, or at most about 97% of the total matrix volume, about 50% to about 97% of the total matrix volume, about 60% to about 97% of the total matrix volume, about 70% to about 97% of the total matrix volume, about 80% to about 97% of the total matrix volume, about 90% to about 97% of the total matrix volume, about 50% to about 95% of the total matrix volume, about 60% to about 95% of the total matrix volume, about 70% to about 95% of the total matrix volume, about 80% to about 95% of the total matrix volume, about 90% to about 95% of the total matrix volume, about 50% to about 90% of the total matrix volume, about 60% to about 90% of the total matrix volume, about 70% to about 90% of the total matrix volume, or about 80% to about 90% of the total matrix volume.

[0173] The porous structure may be a porous matrix, a porous membrane, an ionomer permeable to a certain type of ion, a porous glass frit, an ion-selective membrane, an ion-conducting glass, a polymer membrane, or an ion-conducting membrane. The porous structure may be formed of a microporous material, such as a ceramic or glass frit, a ceramic or glass membrane, or a solid porous substrate, such as a frit or wafer prepared from a polymer or inorganic material. The glass frit may contain, for example, magnesium oxide, calcium oxide, strontium oxide, barium oxide, cesium oxide, sodium oxide, potassium oxide, boron oxide, vanadium oxide, zinc oxide, tellurium oxide, aluminum oxide, silicon dioxide, lead oxide, tin oxide, phosphorus oxide, ruthenium oxide, rhodium oxide, iron oxide, copper oxide, manganese dioxide, molybdenum oxide, niobium oxide, titanium oxide, tungsten oxide, bismuth oxide, zirconium oxide, lithium oxide, antimony oxide, lead borate glass, tin phosphate glass, vanadate glass, or borosilicate glass.

[0174] In some embodiments, the porous structure may include a microporous membrane formed of polysulfone, polyethersulfone, or polyvinylidene fluoride. In some embodiments, the porous structure may be formed of a resin material such as a polyolefin, such as polyethylene (PE), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polypropylene (PP), polytetrafluoroethylene (PTFE), etc. Additionally, hollow fiber membranes in a laminated structure may be used with a non-porous film and a porous film provided to hold the non-porous film therebetween. In some examples, the porous structure can be formed of PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate), silicon, polytetrafluoroethylene, ethylene-tetrafluoroethylene copolymers, polyolefins, polyesters, polycarbonates, biostable polytetrafluoroethylene, polyurethane homopolymers, copolymers, and terpolymers, polypropylene (PP), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), polymethylmethacrylate (PMMA), polyether ether ketone (PEEK), polyurethanes, cellulosic polymers, polysulfones, and block copolymers thereof, including, for example, diblock, triblock, alternating, random, and graft copolymers.

[0175] In some embodiments, the porous structure may be formed of porous silicon dioxide, organosilicate glass (carbon-doped oxide), indium tin oxide (ITO), or low-κ (low dielectric constant) dielectrics including silicon carbon boron nitride (SiCBN), silicon oxycarbonitride (SiOCN), fluorine-doped silicon dioxide, carbon-doped silicon dioxide, diamond-like carbon (DLC), and combinations thereof. Such porous low-κ materials are commercially available for growth using chemical vapor deposition (CVD) under trade names such as Orion™ from Trikon, BDIIx™ from AMAT, and Aurora™ from ASMi. Alternative materials may be deposited by spinning, such materials include SiLK™ from Dow Chemical and LKD™ from JSR. For example, a low-κ porous organosilicate glass may have a dielectric constant of approximately 2.7 and a porosity (defined as the volume of pores divided by the total volume including the pores and the material between the pores) of greater than 10%. For example, porous silicon dioxide may have a porosity of about 15-40%, or about 30-35%. Porous silicon dioxide may be: <100> The porous structure may have a configuration of vertical and horizontal pores according to the crystal orientation of the silicon body. The porous silicon dioxide may be formed from a substrate material, for example, based on porous silicon. In some examples, the porous structure may be formed of a porous material formed by porosification. In some examples, the porous material may have pores with a size or diameter on the order of nanometers, i.e., a nanoporous material. The porous material formed by porosification may be provided with small pores, for example, about 2 nm to about 100 nm. The porous material formed by porosification may be made with an open porosity of more than 30%.In some examples, the porous structure may be formed of a porous material formed by porosification of low-κ materials, including, but not limited to, silicon boron nitride (SiBN), silicon carbon nitride (SiCN), silicon boron carbon nitride (SiBCN), hydrogen silsesquioxane polymer (HSQ), methyl silsesquioxane polymer (MSQ), polyphenylene oligomers, methyl doped silica or SiOx(CH3)y, SiCxOyHy or SiOCH, organosilicate glasses (SiCOH), silicon oxide, boron nitride, and silicon oxynitride.

[0176] The aspects and embodiments described and claimed herein can be understood in light of the above definitions.

[0177] Additional Notes It should be understood that all combinations of the foregoing concepts and further concepts discussed in more detail below (unless such concepts are mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein. It should also be understood that the terms expressly used in this specification and which may also appear in any disclosures incorporated by reference should be given the meaning most consistent with the particular concepts disclosed herein.

[0178] References throughout this specification to "one example," "another example," "an example," etc. mean that a particular element (e.g., a feature, structure, and / or characteristic) described in connection with an example is included in at least one example described herein, and may or may not be present in other examples. Moreover, unless the context clearly dictates otherwise, it should be understood that the described elements with respect to any example may be combined in any suitable manner in the various examples.

[0179] Ranges provided herein should be understood to include the stated range and any value or subrange within that stated range, as if such value or subrange were expressly recited. For example, a range of about 2 nm to about 20 nm should be interpreted to include not only the explicitly recited limits of about 2 nm to about 20 nm, but also individual values, such as about 3.5 nm, about 8 nm, 18.2 nm, etc., and subranges, such as about 5 nm to about 10 nm, etc. Additionally, when "about" and / or "substantially" are used to express a value, this is meant to encompass slight variations (up to ±10%) from the stated value.

[0180] Although several embodiments have been described in detail, it should be understood that the disclosed examples may be modified, and therefore the foregoing description should be considered as non-limiting.

[0181] Although certain specific embodiments are described, these embodiments are presented as examples only and are not intended to limit the scope of the present disclosure. Indeed, the novel methods and systems described herein may be embodied in a variety of other forms. Furthermore, various omissions, substitutions, and modifications in the systems and methods described herein may be made without departing from the spirit of the present disclosure. The appended claims and their equivalents are intended to cover such forms or modifications as fall within the scope and spirit of the present disclosure.

[0182] It shall be understood that features, materials, properties, or groups described in connection with a particular aspect or embodiment are applicable to any other aspect or embodiment described in this section or elsewhere in this specification, unless incompatible therewith. All of the steps of any method or process disclosed herein (including any accompanying claims, abstract and drawings), and / or so disclosed, may be combined in any combination, except where combinations of at least some of such features and / or steps are mutually exclusive. Protection is not limited to the details of any preceding embodiment. Protection extends to any novel one, or any novel combination, of features disclosed herein (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination of steps of any method or process so disclosed.

[0183] Moreover, certain features that are described herein in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Furthermore, although features may be described above as functioning in certain combinations, one or more features from a claimed combination may, in some cases, be deleted from the combination, and the combination may be claimed as a subcombination or a variation of the subcombination.

[0184] Furthermore, although operations may be shown in the figures or described in the specification in a particular order, such operations do not require that the particular order shown or sequential order be performed, or that all operations be performed, to achieve desirable results. Other operations not shown or described may be incorporated into the example methods and processes. For example, one or more additional operations may be performed before, after, simultaneously with, or between any of the described operations. Furthermore, operations may be rearranged or reordered in other implementations. Those skilled in the art will appreciate that in some embodiments, the actual steps taken in the illustrated and / or disclosed processes may differ from those shown in the figures. Depending on the embodiment, certain steps described above may be removed or others may be added. Furthermore, features and attributes of the specific embodiments disclosed above may be combined in different ways to form additional embodiments, all of which are within the scope of the present disclosure. Also, it should be understood that the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and that the described components and systems may generally be integrated together in a single product or packaged within multiple products. For example, any of the components for the energy storage systems described herein may be provided separately or integrated together (e.g., packaged together or attached together) to form an energy storage system.

[0185] For purposes of this disclosure, certain aspects, advantages, and novel features have been described herein. Not necessarily all such advantages may be achieved in accordance with a particular embodiment. Thus, for example, one skilled in the art will recognize that the disclosure may be embodied or performed in a manner that achieves one advantage or group of advantages taught herein without necessarily achieving other advantages that may be taught or suggested herein.

[0186] Unless otherwise stated, conditional language such as "can," "could," "may," or "might," unless otherwise stated or as understood within the context in which it is used, is intended to convey that, in general, a particular embodiment includes certain features, elements, and / or steps and other embodiments do not. Thus, such conditional language is not intended to imply that features, elements, and / or steps are in any way required by one or more embodiments, or that one or more embodiments necessarily include logic that determines whether those features, elements, and / or steps should be included or performed in any particular embodiment, with or without user input or prompting.

[0187] Conjunctive language, such as the phrase "at least one of X, Y, and Z," is understood in the context as it is commonly used, unless otherwise noted, to convey that an item, term, etc. can be either X, Y, or Z. Thus, such conjunctive language is not generally intended to imply that a particular embodiment requires the presence of at least one of X, at least one of Y, and at least one of Z.

[0188] As used herein, language of degree, such as the terms "approximately," "about," "generally," and "substantially," denotes a value, amount, or characteristic that approaches a specified value, amount, or characteristic that still performs a desired function or achieves a desired result.

[0189] The scope of the present disclosure is not intended to be limited by the specific disclosure of preferred embodiments in this section or elsewhere herein, but may be defined by the claims presented in this section or elsewhere herein, or presented in the future. Claim language is to be interpreted broadly based on the language employed in the claims, and is not to be limited to the embodiments herein or during the prosecution of this application, which embodiments are to be construed as non-exclusive.

Claims

1. A device, comprising: a central well comprising a fluid tunnel; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a second nanoscale opening disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the second nanoscale opening, the FET comprising: a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having an upper surface fluidly exposed to the central well; A device wherein the central well fluidly connects the syswell to the transwell, and the fluid tunnel extends through the channel.

2. A device, comprising: a central well comprising a fluid tunnel; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a second nanoscale opening disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the second nanoscale opening, the FET comprising: a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having an upper surface and a lower surface, the surfaces being fluidly exposed to the central well; A device wherein the central well fluidly connects the syswell to the transwell, and the fluid tunnel extends through the channel.

3. A device, comprising: a central well comprising a fluid tunnel; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a porous structure disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the porous structure, the FET comprising: a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having an upper surface fluidly exposed to the central well; A device wherein the central well fluidly connects the syswell to the transwell, and the fluid tunnel extends through the channel.

4. A device, comprising: a central well comprising a fluid tunnel; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a second nanoscale opening disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the second nanoscale opening, the FET comprising: a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having an upper surface fluidly exposed to the central well; A device wherein the central well fluidly connects the syswell to the transwell, and the fluid tunnel does not extend through the channel.

5. A device, comprising: a central well comprising a fluid tunnel; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a second nanoscale opening disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the second nanoscale opening, the FET comprising: a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having an upper surface and a lower surface, the surfaces being fluidly exposed to the central well; A device wherein the central well fluidly connects the syswell to the transwell, and the fluid tunnel does not extend through the channel.

6. A device, comprising: a central well comprising a fluid tunnel; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a porous structure disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the porous structure, the FET comprising: a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer having an upper surface fluidly exposed to the central well; A device wherein the central well fluidly connects the syswell to the transwell, and the fluid tunnel does not extend through the channel.

7. The device of any one of claims 1 to 6, wherein the FET is a nanowire transistor.

8. 8. The device of claim 7, wherein the channel has a length along a direction from the source to the drain, a height along a direction from the cis electrode to the trans electrode, and a width along a direction at least substantially orthogonal to both the length and the height, the length being at least about 10 times the width or the height.

9. The device of claim 8 , wherein an intersection between the fluid tunnel and the channel in a plane defined by the length and width is disk-shaped.

10. The device of any one of claims 1 to 6, wherein the FET is a nanosheet transistor.

11. 11. The device of claim 10, wherein the channel has a length along a direction from the source to the drain, a height along a direction from the cis electrode to the trans electrode, and a width along a direction at least partially orthogonal to both the length and the height, wherein the length is at least about 5 times the height and the width is at least about 5 times the height.

12. The device of claim 11 , wherein an intersection between the fluid tunnel and the channel in a plane defined by the length and width is elliptical in shape.

13. 7. The device of claim 1, wherein the FET further comprises a plurality of channels, each of the plurality of channels comprising a gate oxide layer having upper and lower surfaces fluidly exposed to the central well, and the fluid tunnel extending through each of the plurality of channels.

14. The device of claim 3 or 6, wherein the porous structure comprises a SiCOH film.

15. The device of any one of claims 1 to 6, further comprising a membrane positioned between the cis well and the central well, the first nanoscale opening extending through the membrane.

16. The device of claim 15 , wherein the membrane is selected from the group consisting of lipids and biomimetic equivalents of lipids.

17. 16. The device of claim 15, wherein the first nanoscale opening extends through a polynucleotide nanopore, a polypeptide nanopore, or a carbon nanotube disposed in the membrane.

18. The device of claim 15 , wherein the membrane is a synthetic membrane and the first nanoscale opening is a solid-state nanopore.

19. The device of any one of claims 1 to 6, wherein the device comprises a nanopore sequencer.

20. The device of any one of claims 1 to 6, wherein the gate oxide layer has a thickness of about 1 to about 10 nm.

21. The device of any one of claims 1 to 6, wherein the gate oxide layer has a thickness of about 2 to about 4 nm.

22. The device of claim 1 , 2 or 3 , wherein the gate oxide layer further comprises a vertical surface fluidically exposed to the fluid tunnel.

23. 20. An array of nanopore sequencers according to claim 19, comprising: each of the plurality of nanopore sequencers shares a common cis electrode and a common trans electrode; or each of the plurality of nanopore sequencers has a separate cis electrode and a separate trans electrode; or each of the plurality of nanopore sequencers shares a common cis electrode and has a separate trans electrode; or An array, wherein each of the plurality of nanopore sequencers has a separate cis electrode and shares a common trans electrode.

24. A method of using a device according to any one of claims 1 to 6, said method comprising the steps of: introducing an electrolyte into each of the syswells, the transwells, the central well, and the fluidic tunnels of the device; applying a voltage bias between the cis electrode and the trans electrode, where an electrical resistance of the first nanoscale opening changes in response to an identity of a base in a polynucleotide at the first nanoscale opening and where a potential of the electrolyte in the fluid tunnel changes in response to the variation in electrical resistance of the first nanoscale opening; and measuring a response of the FET at the first nanoscale opening as a function of the base in the polynucleotide to identify a base in the polynucleotide.

25. Measuring the response of the FET the source-drain current, or a potential at the source, the drain, or both the source and the drain; or the resistance of the channel, or 25. The method of claim 24, comprising measuring any combination thereof.

26. A device, comprising: A central well; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a second nanoscale opening disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the second nanoscale opening, the FET comprising: a source, a drain, and a channel connecting said source to said drain, said source, drain, and channel being vertically stacked, said channel comprising a gate oxide layer having a vertical surface fluidically exposed to said central well; the central well fluidly connects the syswell to the transwell; A device wherein the perpendicular direction is from the cis electrode to the trans electrode.

27. A device, comprising: A central well; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a porous structure disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the porous structure, the FET comprising: a source, a drain, and a channel connecting said source to said drain, said source, drain, and channel being vertically stacked, said channel comprising a gate oxide layer having a vertical surface fluidically exposed to said central well; the central well fluidly connects the syswell to the transwell; A device wherein the perpendicular direction is from the cis electrode to the trans electrode.

28. 28. The device of claim 26 or 27, wherein the FET is a nanowire transistor.

29. 28. The device of claim 26 or 27, wherein the FET is a nanosheet transistor.

30. 28. The device of claim 26 or 27, wherein the FET further comprises a plurality of channels, each of the plurality of channels comprising a gate oxide layer having two additional vertical surfaces fluidly exposed to the central well.

31. The device of claim 27 , wherein the porous structure comprises a SiCOH film.

32. 28. The device of claim 26 or 27, further comprising a membrane positioned between the cis well and the central well, the first nanoscale opening extending through the membrane.

33. 33. The device of claim 32, wherein the membrane is selected from the group consisting of lipids and biomimetic equivalents of lipids.

34. 33. The device of claim 32, wherein the first nanoscale opening extends through a polynucleotide nanopore, a polypeptide nanopore, or a carbon nanotube disposed in the membrane.

35. 33. The device of claim 32, wherein the membrane is a synthetic membrane and the first nanoscale opening is a solid-state nanopore.

36. The device of claim 26 or 27, wherein the gate oxide layer has a thickness of about 1 to about 10 nm.

37. The device of claim 26 or 27, wherein the gate oxide layer has a thickness of about 2 to about 4 nm.

38. The device of claim 26 or 27, wherein the device comprises a nanopore sequencer.

39. 39. An array of nanopore sequencers according to claim 38, comprising: each of the plurality of nanopore sequencers shares a common cis electrode and a common trans electrode; or each of the plurality of nanopore sequencers has a separate cis electrode and a separate trans electrode; or each of the plurality of nanopore sequencers shares a common cis electrode and has a separate trans electrode; or An array, wherein each of the plurality of nanopore sequencers has a separate cis electrode and shares a common trans electrode.

40. 28. A method of using a device according to claim 26 or 27, said method comprising: introducing an electrolyte into each of the syswell, the transwell, and the central well of the device; applying a voltage bias between the cis electrode and the trans electrode, where an electrical resistance of the first nanoscale opening changes in response to an identity of a base in a polynucleotide at the first nanoscale opening and where a potential of the electrolyte in the central well changes in response to the variation in electrical resistance of the first nanoscale opening; and measuring a response of the FET at the first nanoscale opening as a function of the base in the polynucleotide to identify a base in the polynucleotide.

41. Measuring the response of the FET source-drain current, or a potential at the source, the drain, or both the source and the drain; or the resistance of the channel, or 41. The method of claim 40, comprising measuring any combination thereof.

42. A device, comprising: A central well; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a second nanoscale opening disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the second nanoscale opening, the FET comprising: a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer operatively connected to a metal structure; the central well fluidly connects the syswell to the transwell; The device, wherein the metal structure has at least one surface fluidly exposed to the central well.

43. A device, comprising: A central well; a cis-well associated with a cis-electrode, the cis-well having a first nanoscale opening disposed between the cis-well and the central well; a transwell associated with a trans electrode, the transwell having a porous structure disposed between the transwell and the central well; a field effect transistor (FET) positioned between the first nanoscale opening and the porous structure, the FET comprising: a source, a drain, and a channel connecting the source to the drain, the channel comprising a gate oxide layer operatively connected to a metal structure; the central well fluidly connects the syswell to the transwell; The device, wherein the metal structure has at least one surface fluidly exposed to the central well.

44. 44. The device of claim 42 or 43, wherein the gate oxide layer is not fluidly exposed.

45. 44. The method of claim 42 or 43, wherein the at least one fluidly exposed surface of the metal structure is formed of a corrosion resistant material.

46. 44. The device of claim 42 or 43, wherein the metallic structure has at least one partially vertical surface, at least two partially vertical surfaces, at least one partially horizontal surface, at least two partially horizontal surfaces, or any combination thereof, fluidly exposed to the central well, the vertical direction being from the cis electrode to the trans electrode, and the horizontal direction being orthogonal to the vertical direction.

47. 44. The device of claim 42 or 43, wherein the metal structure has at least one cup-shaped substructure fluidly exposed to the central well.

48. 44. The device of claim 42 or 43, wherein the portion of the metal structure fluidly exposed to the central well comprises at least one hole or opening.

49. 44. The device of claim 42 or 43, wherein the portion of the metal structure fluidly exposed to the central well comprises at least two holes or openings.

50. 44. The device of claim 42 or 43, wherein the FET is a nanowire transistor.

51. 51. The device of claim 50, wherein the channel has a length along a direction from the source to the drain, a height along a direction at least partially orthogonal to the length, and a width along a direction at least partially orthogonal to both the length and the height, the length being at least about 10 times the width or the height.

52. 44. The device of claim 42 or 43, wherein the FET is a nanosheet transistor.

53. 53. The device of claim 52, wherein the channel has a length along a direction from the source to the drain, a height along a direction at least substantially orthogonal to the length, and a width along a direction at least partially orthogonal to both the length and the height, wherein the length is at least about 5 times the height and the width is at least about 5 times the height.

54. The device of claim 43 , wherein the porous structure comprises a SiCOH film.

55. 44. The device of claim 42 or 43, further comprising a membrane positioned between the cis well and the central well, the first nanoscale opening extending through the membrane.

56. 56. The device of claim 55, wherein the membrane is selected from the group consisting of lipids and biomimetic equivalents of lipids.

57. 56. The device of claim 55, wherein the first nanoscale opening extends through a polynucleotide nanopore, a polypeptide nanopore, or a carbon nanotube disposed in the membrane.

58. 56. The device of claim 55, wherein the membrane is a synthetic membrane and the first nanoscale opening is a solid-state nanopore.

59. The device of claim 42 or 43, wherein the gate oxide layer has a thickness of about 1 to about 10 nm.

60. The device of claim 42 or 43, wherein the gate oxide layer has a thickness of about 2 to about 4 nm.

61. The device of claim 42 or 43, wherein the device comprises a nanopore sequencer.

62. 62. An array of nanopore sequencers according to claim 61, comprising: each of the plurality of nanopore sequencers shares a common cis electrode and a common trans electrode; or each of the plurality of nanopore sequencers has a separate cis electrode and a separate trans electrode; or each of the plurality of nanopore sequencers shares a common cis electrode and has a separate trans electrode; or An array, wherein each of the plurality of nanopore sequencers has a separate cis electrode and shares a common trans electrode.

63. 44. A method of using a device according to claim 42 or 43, said method comprising: introducing an electrolyte into each of the syswell, the transwell, and the central well of the device; applying a voltage bias between the cis electrode and the trans electrode, where an electrical resistance of the first nanoscale opening changes in response to an identity of a base in a polynucleotide at the first nanoscale opening and where a potential of the electrolyte in the central well changes in response to the variation in electrical resistance of the first nanoscale opening; and measuring a response of the FET at the first nanoscale opening as a function of the base in the polynucleotide to identify a base in the polynucleotide.

64. Measuring the response of the FET the source-drain current, or a potential at the source, the drain, or both the source and the drain; or the resistance of the channel, or 64. The method of claim 63, comprising measuring any combination thereof.

65. 64. The method of claim 63, wherein an electrochemical reaction does not occur at the at least one fluidly exposed surface of the metal structure.

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