Device with field effect transistor

Abstract

Apparatuses and methods of using the apparatuses are disclosed that can provide scalability, increased sensitivity, and reduced noise for sequencing polynucleotides. Examples of apparatuses include biological or solid-state nanopores, field effect transistor (FET) sensors with improved gate controllability over a channel, and porous structures.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 047743, filed July 2, 2020, and U.S. Provisional Application No. 63 / 200868, filed March 31, 2021, the entire contents of each of which are incorporated herein by reference. Background Technology

[0003] Various polynucleotide sequencing technologies involve conducting a large number of controlled reactions on a support surface or in a predefined reaction chamber. The controlled reactions can then be observed or detected, and subsequent analysis can help identify the characteristics of the polynucleotides involved in the reaction.

[0004] Some of these polynucleotide sequencing technologies utilize nanopores, which can provide pathways for ion currents. For example, as a polynucleotide passes through a nanopore, it influences the current flowing through the nanopore. Each passing nucleotide or series of nucleotides through the nanopore generates a characteristic current. These characteristic currents flowing through the polynucleotide can be recorded to determine the polynucleotide's sequence.

[0005] Figure 1A A prior art nanopore sequencing device 1110, as shown in PCT Publication WO 2019 / 160925, is illustrated. The prior art nanopore sequencing device 1110 includes a cis-well 1114 associated with a cis-electrode 1130, an inverse-well 1116 associated with an inverse-electrode 1134, and a field-effect transistor (FET) 1122 located between the cis-well 1114 and the inverse-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 inverse-well 1116 includes a second cavity 1117. A fluid tunnel 1121 extends from the first cavity 1115 through the FET 1122 to the inverse-well 1116. An electrolyte 1120 is disposed in the cis-well 1114, the first cavity 1115, and the inverse-well 1116.

[0006] Between the cis-trap 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 connects the electrolyte from the cis-trap 14 and electrically connects it to the first cavity 1115. The first nanoscale opening 1123 has an inner diameter 1123'. When the polynucleotide 1129 passes through the first nanoscale opening 1123, the sequence of the polynucleotide can be determined by measuring the voltage change of the FET sensor 1122. A second nanoscale opening 1125 within the base substrate 1162' fluidly connects the fluid tunnel 1121 and the second cavity 1117, wherein the second nanoscale opening 1125 has an inner diameter 1125'.

[0007] Metal interconnects 1164' and 1166' are electrically connected to the source 1150 and drain 1152 of the FET 1122. A relatively thick interlayer dielectric 1168, typically thicker than about 50 nm, surrounds the channel 1154 and the upper and lower surfaces of the FET sensor 1122 to form a fluid tunnel 1121. The FET sensor 1122 is electrically connected to the electrolyte 1120 at boundary 1156, where the channel 1154 is closest to the fluid tunnel 1121. As shown, the thickness of the interlayer dielectric 1168 above or below the channel 1154 can be about three times or more the thickness of the channel 1154 of the FET 1122. Summary of the Invention

[0008] This article provides examples of devices for sequencing polynucleotides and methods for using those devices. One example of such a device is a nanopore device. Specifically, examples include devices with field-effect transistor (FET) sensors and porous structures.

[0009] The systems, apparatuses, kits, and methods disclosed herein each have several aspects, none of which individually bears responsibility for their desired properties. Without limiting the scope of the claims, some prominent features will now be briefly discussed. Many other examples are also contemplated, including those with fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. Components, aspects, and steps may also be arranged and ordered differently. After considering this discussion, and especially after reading the section entitled "Detailed Description," one will understand how the features of the apparatuses and methods disclosed herein provide advantages over other known apparatuses and methods.

[0010] One example is an apparatus comprising: an intermediate well including a fluid tunnel; a cis-well associated with a cis-electrode, wherein a first nanoscale opening is disposed between the cis-well and the intermediate well; an inverse well associated with an inverse electrode, wherein a second nanoscale opening is disposed between the inverse well and the intermediate well; and a field-effect transistor (FET) located between the first nanoscale opening and the second nanoscale opening. In this example, the FET includes: a source, a drain, and a channel connecting the source to the drain, wherein the channel includes a gate oxide layer having a surface fluidly exposed above the intermediate well, wherein the intermediate well fluidly connects the cis-well to the inverse well. In some embodiments, the fluid tunnel extends through the channel. In alternative embodiments, the fluid tunnel is offset from the FET channel (i.e., does not extend through the FET channel).

[0011] Another example is a device comprising: an intermediate well including a fluid tunnel; a cis-well associated with a cis-electrode, wherein a first nanoscale opening is disposed between the cis-well and the intermediate well; an inverse well associated with an inverse electrode, wherein a second nanoscale opening is disposed between the inverse well and the intermediate well; and a field-effect transistor (FET) located between the first and second nanoscale openings, the FET including: a source, a drain, and a channel connecting the source to the drain, wherein the channel includes a gate oxide layer having an upper surface and a lower surface fluidly exposed to the intermediate well, wherein the intermediate well fluidly connects the cis-well to the inverse well. In some embodiments, the fluid tunnel extends through the channel. In alternative embodiments, the fluid tunnel is offset from the FET channel (i.e., does not extend through the FET channel).

[0012] Another example is a device comprising: an intermediate well including a fluid tunnel; a cis-well associated with a cis-electrode, wherein a first nanoscale opening is disposed between the cis-well and the intermediate well; an inverse well associated with an inverse electrode, wherein a porous structure is disposed between the inverse well and the intermediate well; and a field-effect transistor (FET) located between the first nanoscale opening and the porous structure, the FET including: a source, a drain, and a channel connecting the source to the drain, wherein the channel includes a gate oxide layer having a fluidly exposed upper surface of the intermediate well, wherein the intermediate well fluidly connects the cis-well to the inverse well. In some embodiments, the fluid tunnel extends through the FET channel. In alternative embodiments, the fluid tunnel is offset from the FET channel (i.e., does not extend through the FET channel).

[0013] Another example is a method of using any of the aforementioned devices in the method, the method comprising: introducing an electrolyte into each of the cis-trap, trans-trap, intermediate trap, and fluid tunnel of the device; applying a bias voltage between the cis-electrode and the trans-electrode, wherein the resistance of the first nanoscale opening varies in response to the base identity in the polynucleotide at the first nanoscale opening, and wherein the potential (V) of the electrolyte in the fluid tunnel is... M The response of the FET changes in response to a change in the resistance of the first nanoscale opening; and the response of the FET is measured as a base function in the polynucleotide at the first nanoscale opening to identify the bases in the polynucleotide.

[0014] It should be understood that any features of the apparatus and / or array disclosed herein can be combined in any desired manner and / or configuration. Furthermore, it should be understood that any features of the method using the apparatus can be combined in any desired manner. Moreover, it should be understood that any combination of features of this method and / or apparatus and / or array can be used together, and / or can be combined with any example of the examples disclosed herein. Further, it should be understood that any feature or combination of features of any apparatus in the apparatus and / or any array in the array and / or any method in the method can be combined in any desired manner, and / or can be combined with any example of the examples disclosed herein.

[0015] It should be understood that all combinations of the foregoing concepts and the additional concepts discussed in more detail below are contemplated as part of the inventive subject matter disclosed herein and can be used to achieve the benefits and advantages described herein. Attached Figure Description

[0016] The features of this disclosure will become apparent from the following detailed description and accompanying drawings, in which similar reference numerals correspond to similar but possibly different parts. For the sake of brevity, reference numerals or features having the functions previously described may be described in conjunction with or without conjunction with other accompanying drawings in which they appear.

[0017] Figure 1A This is a cross-sectional side view of a current nanopore sequencing device.

[0018] Figure 1B It shows the result of Figure 1A A schematic circuit diagram of the resistor provided by existing nanopore sequencing devices.

[0019] Figure 2A This is a cross-sectional side view based on an example nanopore sequencing device.

[0020] Figure 2B Is Figure 2A A top view of a cross-section taken from line 3-3 of the nanopore sequencing device.

[0021] Figure 2B' Is Figure 2A A top view of a cross-section taken from line 3'-3' of the nanopore sequencing device.

[0022] Figure 3A A cross-sectional side view of an alternative example of a nanopore sequencing device based on an example is shown.

[0023] Figure 3B Is Figure 3A A top-view cross-section of the nanopore sequencing device and FET sensor taken along line 3-3.

[0024] Figure 3C Is Figure 3A A top view of the cross-section taken along line 3'-3' of the nanopore sequencing device and FET sensor.

[0025] Figure 3D In a similar Figure 3A An alternative example of a cross-sectional top view taken from line 3-3 of a nanopore sequencing device, but a broader example with a FET sensor.

[0026] Figure 3E In a similar Figure 3A An alternative example of a cross-sectional top view taken from line 3'-3' of a nanopore sequencing device, but a broader example with a FET sensor.

[0027] Figure 4A This is another cross-sectional side view of an alternative example of a nanopore sequencing device.

[0028] Figure 4B Is Figure 4A A top view of a cross-section taken from line 3-3 of the nanopore sequencing device.

[0029] Figure 4B' Is Figure 4A A top view of a cross-section taken from line 3'-3' of the nanopore sequencing device.

[0030] Figure 5A This is a cross-sectional side view of yet another alternative example of a nanopore sequencing device.

[0031] Figure 5B Is Figure 5A A top view of a cross-section taken from line 3-3 of the nanopore sequencing device.

[0032] Figure 5B' Is Figure 5A A top view of a cross-section taken from line 3'-3' of the nanopore sequencing device.

[0033] Figure 6This is a cross-sectional side view of another exemplary alternative example of a nanopore sequencing device.

[0034] Figure 7A This is a cross-sectional side view of yet another exemplary alternative example of a nanopore sequencing device with an offset opening.

[0035] Figure 7B Is Figure 5A A top-view cross-section of the offset opening is shown on line 3-3 of the nanopore sequencing device.

[0036] Figure 7B' Is Figure 5A A top-view cross-section of the offset opening is shown, taken from line 3'-3' of the nanopore sequencing device.

[0037] Figure 8 This is a cross-sectional side view of another exemplary alternative example of a nanopore sequencing device with a vertical field-effect transistor.

[0038] Figure 9 This is a cross-sectional side view of yet another exemplary alternative example of a nanopore sequencing device with a field-effect transistor having a non-Radida metal electrode. Detailed Implementation

[0039] All patents, applications, published applications and other publications mentioned herein are incorporated herein by reference, and their entire contents are incorporated herein by reference. If a term or phrase is used herein in a manner contrary to or otherwise inconsistent with its definition set forth in the patents, applications, published applications and other publications incorporated herein by reference, its use herein shall prevail over the definition incorporated herein by reference.

[0040] One example relates to a sequencing device including a field-effect transistor (FET) sensor having a channel disposed between the source and drain of the FET sensor. While the sequencing device is described as a nanopore device in many instances herein, the device need not be a nanopore device, and other configurations are possible. In one example, the channel has an upper surface, a lower surface, or both exposed to an electrolyte within the device. The exposed upper and / or lower surface of the FET sensor provides an increased surface area for electrical contact with the electrolyte, which 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, thus providing a multifactorial enhancement to the signal-to-noise ratio (SNR) when measuring nucleic acid sequences in contact with the nanopore.

[0041] In one example, the nanopore sequencing system utilizes a FET sensor with a gate-all-ring (GAA) transistor to further increase the device's signal-to-noise ratio. This GAA technology allows the FET sensor to have not only an upper surface exposed to the electrolyte but also a lower surface exposed to the electrolyte. See below for reference. Figure 4A Describe more information about this structure. In one embodiment, one or more all-ring gate transistors of the nanopore sequencing system may include upper and lower surfaces of a source-drain channel exposed to the electrolyte, such as... Figure 4A , Figure 4B and Figure 4B' As shown. In another embodiment, one or more all-ring gate transistors of the nanopore sequencing system may include multiple source-drain channels exposed on the upper and lower surfaces of the electrolyte, such as... Figure 6 As shown. In yet another embodiment, one or more all-ring gate transistors of the nanopore sequencing system may include vertical transistors, such as... Figure 8 As shown.

[0042] In another example, the FET is not in direct contact with the electrolyte. Instead, as... Figure 9 The non-Radidatic metal electrode shown is exposed to the electrolyte and transmits the detected signal to the sensing FET. This configuration allows for significant simplification of the manufacturing process and better compatibility with conventional semiconductor process flows.

[0043] In another example, solid-state nanoporous structures can be replaced by porous structures, as discussed in more detail below. Such porous structures can be more easily integrated into semiconductor manufacturing processes.

[0044] As used herein, the term "exposed to electrolyte" does not necessarily mean that a component is in direct contact with the electrolyte. For example, a FET sensor or the channel of a FET sensor exposed to an electrolyte may include a relatively thin layer of insulator between the sensor or channel and the electrolyte. For example, in one example, the channel portion of a FET sensor located between the source and drain may be covered by a relatively thin layer of gate oxide (e.g., a thermally grown silicon dioxide layer), and the channel having its gate oxide is referred to as "exposed to electrolyte." Alternatively, the thin layer of insulator may be formed of a high-k dielectric, such as HfO2, Al2O3, silicon oxynitride, Si3N4, TiO2, Ta2O5, Y2O3, La2O3, ZrO2, ZrSiO4, barium strontium titanate, lead zirconate titanate, or ZrSiO4. x O y or ZrAl x O yThe thickness of the gate oxide layer may be about 10 nm, or in other examples, less than about 9 nm, about 8 nm, about 7 nm, about 6 nm, about 5 nm, about 4 nm, about 3 nm, about 2 nm, or about 1 nm, and is still within the examples described herein.

[0045] Electrical operation of nanopore sequencing devices

[0046] Now for reference Figure 1B The diagram shows an equivalent circuit diagram of a nanopore device (e.g., the nanopore devices shown in Figures 2 through 7). An electrolyte is introduced into each of the cis-trap, trans-trap, intermediate trap, and fluid tunnel. A voltage difference V is applied between the cis and trans electrodes. In some examples, a polynucleotide is driven through a first nanoscale opening of the first nanopore (e.g., a protein nanopore). In alternative examples, the polynucleotide does not pass through the first nanopore, but the labeled nucleotide is incorporated by a polymerase acting on the polynucleotide. In some embodiments, single-stranded polynucleotides, double-stranded polynucleotides, tags or labels of incorporated nucleotide bases, or other representatives of incorporated nucleotide bases, and any combination thereof, can pass through the first nanopore. In some embodiments, the tags or labels of incorporated nucleotide bases can be detached or dissociated from the polynucleotide, and such tags or labels can pass through the first nanopore with or without the polynucleotide passing through it. Examples are not limited to how polynucleotides communicate with nanopores to induce signal generation in a nanopore sequencing device. The resistance R of the first nanoscale opening is shown. 蛋白质 The resistance of the first nanoscale opening changes in response to the identity of the base at the first nanoscale opening, for example, when the base of the polynucleotide passes through the first nanoscale opening, or when the labeled nucleotide is incorporated by a polymerase acting on the polynucleotide. Thus, different labels of the labeled nucleotides change the resistance of the first nanoscale opening.

[0047] In the example, the second nanoscale opening of the second nanopore (e.g., a solid nanopore) has a fixed or substantially fixed resistance R. 孔 The potential of the electrolyte in the fluid tunnel (represented as the voltage divider point M in Figure 1C) responds to the resistance R of the first nanoscale opening. 蛋白质 The resistance changes with the resistance of the FET. Therefore, measuring the response of the FET as a change in resistance within the first nanometer-scale opening allows for the determination of the resistance within the first nanometer-scale opening, and this information can be used to identify bases in polynucleotides.

[0048] During nanopore sequencing operations, applying a potential (i.e., a voltage difference V) across the first nanopore forces nucleotides, along with charged anions, to translocate through the first nanoscale opening. Depending on the bias voltage, the nucleotide can transport from the cis-well to the intermediate well, or vice versa. As the nucleotide translocates through the first nanoscale opening, the current across membrane 24 changes due to base-dependent blockage, for example, at the constriction. A FET sensor can be used to measure the signal from this current change. Examples of measuring the FET response 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 bases at the first nanoscale opening.

[0049] During operation, the measured voltage range can be selected from approximately -0.1V to above approximately 0.1V, approximately -0.5V to above approximately 0.5V, approximately -1V to above approximately 1V, approximately -1.5V to above approximately 1.5V, approximately -2.0V to above approximately 2.0V, approximately -3.0V to above approximately 3.0V, and approximately -5.0V to above approximately 5.0V. Typically, the applied voltage polarity causes negatively charged nucleic acids to be electrophoretically driven toward the reverse electrode. In some cases, the voltage can be reduced or the polarity reversed to facilitate proper functioning of the device. In a non-limiting example, the resistance R of the first nanometer-scale opening... 蛋白质 The resistance can range from approximately 0.5 gigahertz ohms (GΩ) to approximately 1 GΩ. The resistance R of the second nanometer-scale opening... 孔 It can be approximately 50 megaohms (MΩ). In one example, R 蛋白质 It varies as a function of the bases of the polynucleotide at the first nanoscale opening.

[0050] The voltage at point M of the voltage divider varies with R. 蛋白质 The resistance R of the second nanoscale opening, which can be formed in a solid-state nanopore, changes and acts as the gate potential of the FET. 孔 It is fixed or at least substantially fixed, and 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 is regulated based on the identity of the bases in the polynucleotide. 蛋白质 Alternatively, the resistance R of the first nanoscale opening is adjusted based on the identity of the labeled nucleotide incorporated by a polymerase acting on the polynucleotide. 蛋白质 Resistance R 蛋白质 It can be relatively large and typically varies by 30%–40% as a function of different polynucleotide bases at the first nanoscale opening. In other examples, the resistance R 蛋白质The resistance R can vary between approximately 0.001% and approximately 1%, approximately 1% and approximately 5%, approximately 5% and approximately 20%, approximately 20% and approximately 40%, approximately 40% and approximately 60%, or 60% and approximately 100%. A second nanometer-scale opening with a larger size than the first nanometer-scale opening can also be used. 孔 With R 蛋白质 It can be about 10 times lower. This is because the function of the second nanometer-scale opening is to provide a fixed resistance R in the voltage divider. 孔 (But without reading the current associated with the first nanometer-scale opening), so the second nanometer-scale opening may not need to be atomically precise.

[0051] Figure 1B The equivalent circuit shown is a voltage divider, where the potential at point M is the potential of the electrolyte in the fluid tunnel. This potential is the equivalent gate potential of the FET and establishes its operating point. When the potential V at point M... M As the base identity of the polynucleotide changes, the current flowing through the FET (source-drain current) changes, thereby providing a measurement of the current flowing through the first nanoscale opening, and thus a measurement of the polynucleotide base identity. In some embodiments, the equivalent circuit of the nanopore device satisfies the following equation:

[0052] The electric potential V at point M M- Given from the following:

[0053] V M =DV (1)

[0054] in

[0055]

[0056] It is the voltage divider ratio and V is the forward and reverse bias voltage.

[0057] The signal that drives the FET sensor to respond is δV M Its potential V M The base composition of the polynucleotide at the first nanoscale opening changes. From the above equation, the following relationship can be derived:

[0058] δV M =VδD (3)

[0059] Where δD is the voltage divider ratio as a function of the bases of the polynucleotide at the first nanoscale opening.

[0060] signal δV M This may exceed the detection limit (LoD) of the FET sensor, i.e., VδD > LoD. Therefore, the sensitivity of the nanopore device 10 increases as LoD decreases, V increases, or δD increases.

[0061] The operating forward and reverse bias voltages V can therefore satisfy:

[0062]

[0063] Example

[0064] Figure 2A An example of a nanopore sequencing device with a FET sensor having an increased surface area exposed to electrolytes is shown in the figure. Figure 2A This is a side cross-sectional view of the exemplary device 10A. Figure 2B Is Figure 2A Top view of the cross section taken on line 3-3. Figure 2B' Is Figure 2A Top view of the cross section taken on line 3'-3'.

[0065] Figure 2A , Figure 2B and Figure 2B' The illustrated nanopore sequencing device 10A includes a cis electrode 30A connected to a cis trap 14A. The cis trap 14A has a lower portion including 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, which communicates with a fluid tunnel 21A to a second nanoscale opening 25A disposed in a narrower region 17A between the fluid tunnel 21A and an inverse trap 16A at the lower portion of the device 10A. As shown, the second nanoscale opening is formed in a substrate material 62A. The first nanopore 18A provides a fluid channel for an electrolyte 20A to pass between the cis trap 14A and an intermediate trap 15A. The fluid tunnel 21A provides a fluid channel for the electrolyte to pass through the second nanoscale opening 25A from the intermediate trap 15A and reach the inverse trap 16A.

[0066] In one example, the cis electrode 30A and the anti electrode 34A are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis electrode and the anti electrode may be in any suitable orientation relative to each other and relative to the nanopore device. The nanopore device 10A also includes a field-effect transistor (FET) sensor 22A located between a first nanoscale opening 23A and a 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 view (… Figure 2B and Figure 2B'As shown, electrolyte 20A is visible within fluid tunnel 21A and extends through channel 54A. Metal interconnects 64A and 66A are electrically connected to the source 50A and drain 52A of FET 22A via etch stop layer 38A. Metal interconnects 64A and 66A transmit data from FET sensor 22A to a control system monitoring FET sensor 22A.

[0067] exist Figure 2A In the example of the nanopore device 10A shown, a thin layer of gate oxide 56A is grown around channel 54A, so that the upper surface 55A of this thin layer is fluidly exposed to electrolyte 20A in intermediate well 15A. Gate oxide 56A may have a vertical surface fluidly 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 between about 1 nm and about 10 nm, or alternatively between about 2 nm and about 4 nm. The thickness of gate oxide 56A is chosen such that at a given potential V M In this case, a sufficiently strong electric field can induce an inversion layer of electrons or holes that form the conductive path at the boundary between the channel 54A and the gate oxide 56A, so as to provide measurable conduction between the source 50A and the drain 52A of the FET 22A.

[0068] In this configuration, the upper surface 55A of the gate oxide 56A of the channel 54A fluidly exposes the channel 54A to the electrolyte in the intermediate well 15A, such as Figure 2B As shown. By providing a large area of ​​channel 54A exposed to electrolyte 20A, the potential V M It has better gate controllability on the 54A channel.

[0069] According to the above relations (2) and (4), assume R 蛋白质 The expected horizontal separation is approximately 10% of the orifice resistance, where the expected base voltage divider ratio D is approximately 0.1, then the variation δD is approximately 0.1 x 0.1 = 0.01. Using a FET sensor with 3mV LoD means...

[0070]

[0071] This high forward and reverse bias V may be incompatible with some options for membrane 24A.

[0072] Reducing the LoD to approximately 0.2 mV reduces the required forward and reverse bias voltage V by approximately 15x (15 times) to approximately 20 mV, which is compatible with typical films. This means that FET sensors with large gate areas would be advantageous. In the prior art... Figure 1AIn the FET sensor shown, only a small portion of channel 1154 is exposed to the voltage change δV. M The gate oxide 56A at the boundary of the fluid tunnel 21A is primarily exposed to voltage variations at the boundary of the fluid tunnel 21A. This is in addition to exposing the channel 54A to voltage variations at the boundary of the fluid tunnel 21A. Figure 2A , Figure 2B and Figure 2B' The structure shown, with its exposed upper surface 55A, significantly increases the FET's exposure to δV. M The sensing area was increased and the LoD (with a ratio of 1 / sqrt(A)) was improved, where A is the area of ​​the channel 54A exposed to the electrolyte 20A.

[0073] 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 etchants with high anisotropy, such as fluorinated reactive ion etching, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar as some non-limiting examples.

[0074] Membrane 24A can be either non-permeable or semi-permeable material. A 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 the example, membrane 24A is selected from the group consisting of lipids and biomimetic equivalents of lipids. In another example, membrane 24A is a synthetic membrane (e.g., a solid membrane, an example of which is silicon nitride), and the first nanoscale opening 23A is located in a solid nanopore extending through membrane 24A. In the example, the first nanoscale opening 23A extends through, for example: a polynucleotide nanopore disposed in the membrane; a peptide nanopore; or a solid nanopore, such as a carbon nanotube.

[0075] In one example, the source, drain, and channel of the FET sensor 22A can be formed of silicon, and the surface of the silicon can be thermally oxidized to form a gate oxide on the channel of the FET sensor 22A.

[0076] The first nanopore 18A can be any of a biological nanopore (e.g., a protein nanopore), a solid nanopore, a hybrid nanopore (e.g., a hybrid protein / solid nanopore), and a synthetic nanopore. In some examples, the nanopore has two open ends and a hollow core or cavity (i.e., a first nanoscale opening) connecting the two open ends. When inserted into a membrane, one of the open ends of the nanopore faces a cis-well, and the other open end of the nanopore faces an intermediate well. In some cases, the open end of the nanopore facing the intermediate well is fluidly connected to a fluid tunnel and may also be aligned with at least a portion of the fluid tunnel. In other cases, the open end of the nanopore facing the intermediate well is fluidly connected to the fluid tunnel but not aligned with the fluid tunnel. The hollow core of the nanopore enables fluid and electrical connections between the cis-well and the intermediate well. The diameter of the hollow core of the nanopore can range from about 1 nm to at most about 1 μm and can vary along the length of the nanopore. In some examples, the open end facing the cis-well may be larger than the open end facing the intermediate well. In other examples, the opening facing the cis-well can be smaller than the opening facing the intermediate well.

[0077] The first nanopore 18A can be directly inserted into the membrane, or the membrane can be formed around the nanopore. In one example, the nanopore can insert itself into the formed lipid bilayer membrane. For example, nanopores in monomeric or polymeric form (e.g., octamer) can insert themselves into the lipid bilayer and assemble into transmembrane pores. In another example, nanopores can be added to the grounded side of the lipid bilayer at a desired concentration, wherein the nanopore inserts itself into the lipid bilayer. In yet another example, the lipid bilayer can be formed across the pores in a polytetrafluoroethylene (PTFE) film and positioned between cis-wells and intermediate wells. Nanopores can be added to grounded cis-compartments and can insert themselves into the lipid bilayer at the region where the PTFE pore is formed. In yet another example, the nanopore can be tethered to a solid support (e.g., silicon, silica, quartz, indium tin oxide, gold, polymers, etc.). Tethering molecules (which can be part of the nanopore itself or can be attached to the nanopore) can attach the nanopore to the solid support. Attachment via tethering molecules can fix individual pores (e.g., between cis-wells and intermediate wells). A lipid bilayer can then be formed around the nanopores.

[0078] In one example, the inner diameter of the second nanoscale opening is at least about twice the inner diameter of the first nanoscale opening. In another example, the inner diameter of the second nanoscale opening is about three times the inner diameter of the first nanoscale opening. In yet another example, the inner diameter of the second nanoscale opening is in the range of about twice to about five times the inner diameter of the first nanoscale opening. In these examples, the area of ​​the second nanoscale opening is in the range of about five to about ten times the area of ​​the first nanoscale opening.

[0079] Furthermore, in the example, the inner diameter of the first nanoscale opening is in the range of about 0.5 nm to about 3 nm, and the inner diameter of the second nanoscale opening 25A is in the range of about 10 nm to about 20 nm. In another example, the inner diameter of the first nanoscale opening 23A is in the range of about 1 nm to about 2 nm, and the inner diameter of the second nanoscale opening 25A is in the range of about 10 nm to about 20 nm. In yet another example, the inner diameter of the first nanoscale opening 23A is in the range of about 1 nm to about 3 nm, and the inner diameter of the second nanoscale opening 25A is in the range of about 2 nm to about 20 nm. The exemplary range of the inner diameter of the first nanoscale opening 23A given above is intended to be the minimum diameter of the nanoscale opening 23A passing through the first nanopore 18A.

[0080] The substrate comprising the nanopore sequencing device array can have many different layouts of the first nanoscale openings on the array, including regular, repeating, and irregular patterns of the nanoscale openings. In one example, the first nanoscale openings can be arranged in a hexagonal grid to achieve close packing and increased density of the devices. Other array layouts may include, for example, linear (i.e., rectangular) layouts, triangular layouts, etc. As an example, the layout or pattern can be an xy format of rows and columns of first nanoscale openings. In some other examples, the layout or pattern can be a repeating arrangement of the first nanoscale openings. In still other examples, the layout or pattern can be a random arrangement of the first nanoscale openings. Patterns may include dots, pillars, bars, swirls, lines, triangles, rectangles, circles, arcs, grids, lattices, diagonals, arrows, squares, and / or crosshairs.

[0081] The layout of nanoscale openings can be characterized relative to the density of the first nanoscale openings (i.e., the number of first nanoscale openings in the defined region of the substrate including the array). For example, the array of first nanoscale openings can be at a density of 1 mm. 2 Approximately 10 first nanometer-scale openings per mm 2 A density exists within the range of approximately 1,000,000 first nanometer-scale openings. The density may also include, for example, at least approximately 10 per mm. 2 Approximately 5,000 per mm 2 Approximately 10,000 per mm 2 Approximately 100,000 per mm 2 Or a higher density. Alternatively or additionally, the density may not exceed approximately 1,000,000 particles per mm. 2 Approximately 100,000 per mm 2 Approximately 10,000 per mm 2 Approximately 5,000 per mm 2 Or less. It should also be understood that the density of the first nanoscale opening in the substrate can be between a lower limit value and an upper limit value selected from the above range.

[0082] The layout of the first nanoscale openings in the array on the substrate can also be characterized by the average pitch, i.e., the distance from the center of the first nanoscale opening to the center of the adjacent first nanoscale opening (center-to-center spacing). The pattern can be regular, such that the coefficient of variation around the average pitch is small, or the pattern can be irregular, in which case the coefficient of variation can be relatively large. In the example, the average pitch can be in the range of about 100 nm to about 500 μm. The average pitch can be, for example, at least about 100 nm, about 5 μm, about 10 μm, about 100 μm or greater. Alternatively or additionally, the average pitch can be, for example, at most about 500 μm, about 100 μm, about 50 μm, about 10 μm, about 5 μm or less. The average pitch of an exemplary array of the device can be between a lower limit value and an upper limit value selected from the lower limit value of the above range. In the example, the array can have a pitch (center-to-center spacing) of about 10 μm. In another example, the array can have a pitch (center-to-center spacing) of about 5 μm. In yet another example, the array can have a pitch (center-to-center spacing) in the range of about 1 μm to about 10 μm.

[0083] As described above, a substrate for sequencing can include an array of nanopore sequencing devices. In one example of a nanopore sequencing device, a trans trap is fluidly connected to a cis trap via an intermediate trap and corresponding second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, there may be a common cis trap and a common trans trap, which are in communication with a portion or all of the nanopore sequencing devices within the array on the substrate. However, it should be understood that the array of nanopore devices may also include a plurality of cis traps fluidly isolated from each other and fluidly connected to one or more corresponding trans traps, which are fluidly isolated from each other and defined within the substrate. Multiple cis traps may be required, for example, to enable the measurement of multiple polynucleotides on a single substrate. In some embodiments, a substrate having an array of nanopore sequencing devices includes a common cis electrode, a common trans electrode, a common cis trap, a common trans trap, and multiple nanopore sequencing devices, such as... Figure 2AThe examples shown include each nanopore sequencing device comprising a FET sensor and a dual-well having a first nanopore and a second nanopore. Each nanopore sequencing device in a plurality of nanopore sequencing devices can individually measure resistance or signal via its associated FET sensor. In other embodiments, each nanopore sequencing device may include a multi-well and a FET sensor having three or more nanopores. 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, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of cis-wells, a plurality of trans-wells, and a plurality of nanopore sequencing devices, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode.

[0084] The cis-trap in a nanopore sequencing device can be a fluid chamber defined by sidewalls attached to a substrate. In some examples, the sidewalls and substrate can be integrally formed such that they are formed from a continuous sheet of material (e.g., glass or plastic). In other examples, the sidewalls and substrate can be separate components connected to each other. In some examples, the sidewalls are photopatternable polymers. In some examples, the cis-trap is formed within a space defined by a cis-electrode, a portion of the substrate, and a membrane. The cis-trap can have any suitable size. In some examples, the cis-trap ranges from about 1 mm × 1 mm to about 3 cm × 3 cm. The cis-electrode (whose inner surface forms a surface of the cis-trap) can be physically attached to the sidewalls. The cis-electrode can be physically attached to the sidewalls, for example, by an adhesive or another suitable fastening mechanism. The interface between the cis-electrode and the sidewalls can seal the upper portion of the cis-trap.

[0085] The inverted trap of a nanopore sequencing device is a fluid chamber defined within a portion of a substrate. The inverted trap may extend through the thickness of the substrate and may have openings at opposite ends of the substrate. In some examples, the inverted trap may have sidewalls defined by the substrate and / or by a gap region of the substrate, a lower surface defined by an inverted electrode, and an upper surface defined by a base structure. Thus, the inverted trap can be formed within a space defined by the inverted electrode, another portion of the substrate and / or the gap region, and the base structure. It should be understood that the upper surface of the inverted trap may include a second nanoscale opening to provide fluid communication with an intermediate trap. In some examples, the second nanoscale opening extends through the base structure. In some examples, the second nanoscale opening may fluidly connect to and face a narrower region of the inverted trap.

[0086] The inverted trap can be a micro-trap (having at least one dimension on the micrometer scale, e.g., up to about 1 μm, but excluding about 1000 μm) or a nano-trap (having a maximum dimension on the nanometer scale, e.g., up to about 10 nm, but excluding 1000 nm). An inverted trap can be characterized by its aspect ratio (e.g., in this example, width or diameter divided by depth or height). In the example, the aspect ratio of the inverted trap can range from about 1:1 to about 1:5. In another example, the aspect ratio of each inverted trap can range from about 1:10 to about 1:50. In the example, the aspect ratio of the inverted trap is about 3.3. The depth / height and width / diameter of the inverted trap can be selected to obtain a desired aspect ratio. The depth / height of each inverted trap can be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or greater. Alternatively or additionally, the depth may 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 inverted trap 16 may be at least about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 10 μm, about 100 μm or greater. Alternatively or additionally, the width / diameter may 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.

[0087] Various techniques can be used to fabricate cis- and inverse-wells, including, for example, photolithography, nanoimprint lithography, stamping, imprinting, molding, and micro-etching. As those skilled in the art will understand, the technique used will depend on the composition and shape of the substrate and sidewalls. In the examples, a cis-well may be defined by one or more sidewalls at the ends of the substrate, and an inverse-well may be defined by the substrate.

[0088] The inverted electrode (whose inner surface is the lower surface of the inverted well) can be physically attached to the substrate. The inverted electrode can be fabricated during substrate formation (e.g., during inverted well formation). Microfabrication techniques that can be used to form the substrate and inverted electrode include photolithography, metal deposition and lift-off, drying and / or spin-coating deposition, etching, etc. The interface between the inverted electrode and the substrate can seal the lower portion of the inverted well.

[0089] Examples of materials used to form the basic 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, besides CVD, include atomic layer deposition (ALD). Examples of suitable material combinations for the basic structure 62A include Si3N4, SiO2, SiC, or Al2O3.

[0090] The cis electrode used depends at least in part on the redox pair in the electrolyte. As examples, the cis electrode can be gold (Au), platinum (Pt), carbon (C) (e.g., graphite, diamond, etc.), palladium (Pd), silver (Ag), copper (Cu), etc. In one example, the cis electrode can be a silver / silver chloride (Ag / AgCl) electrode. In one example, the cis trap is capable of maintaining contact between the electrolyte and a first nanoscale opening. In some examples, the cis trap can be in contact with a nanopore array, and thus capable of maintaining contact between the electrolyte and each nanopore in the array.

[0091] The reverse electrode used depends at least in part on the redox pair in the electrolyte. As examples, the reverse electrode can be gold (Au), platinum (Pt), carbon (C) (e.g., graphite, diamond, etc.), palladium (Pd), silver (Ag), copper (Cu), etc. In one example, the reverse electrode can be a silver / silver chloride (Ag / AgCl) electrode.

[0092] In some examples, in NaCl or KCl solution, the relevant electrochemical half-reaction at the Ag / AgCl electrode is:

[0093] cis (cathode): AgCl + e - →Ag 0 +Cl - ; and trans (anode): Ag 0 +Cl - →AgCl+e - .

[0094] For every unit charge of current, one Cl atom is consumed at the inverting electrode. Although the above discussion is for Ag / AgCl electrodes in NaCl or KCl solutions, it should be understood that it applies to any electrode / electrolyte pair that can be used to transfer current.

[0095] In use, the electrolyte can be filled into cis-trap, intermediate trap, fluid tunnel, narrower region, and trans-trap. In alternative examples, the electrolytes in the cis-trap, intermediate trap, and trans-trap can be different. The electrolyte can be any electrolyte capable of dissociating into counterions (cations and their associated anions). As an example, the electrolyte can be capable of dissociating into potassium cations (K+). + ) or sodium cation (Na) +Electrolytes of this type include potassium cations and associated anions, or sodium cations and associated 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., bicarbonate (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) and sodium phosphate (e.g., NaH2PO4, Na2HPO4 or Na3PO4). As another example, the electrolyte can be any electrolyte capable of dissociating into ruthenium-containing cations (e.g., ruthenium hexamine, such as [Ru(NH3)6)). 2+ Or [Ru(NH3)6] 3+ ), can be used to dissociate into lithium cations (Li). + ), rubidium cations (Rb + ), magnesium cations (Mg + ) or calcium cations (Ca + Electrolytes.

[0096] In an example where multiple nanopore sequencing devices are arrayed on a substrate, each nanopore sequencing device in the array may share a common cis electrode and a common trans electrode. In another example, each nanopore sequencing device shares a common cis electrode but has a different trans electrode. In yet another example, each nanopore sequencing device has a different cis electrode and a different trans electrode. In yet another example, each nanopore sequencing device has a different cis electrode and shares a common trans electrode. As the nanopore device array is scaled, the volume of each trans trap is typically exhausted as a power of the trap size (assuming a constant aspect ratio). In some examples, the array lifetime is approximately or greater than 48 hours, and the typical diameter of the trans trap is approximately or greater than 100 μm.

[0097] Alternative examples

[0098] Figure 3A It shows Figure 2A A variant of the device 10A shown. For example... Figure 3A As shown, the nanopore sequencing device 10B includes... Figure 2A The device shown has similar components. However, Figure 3A The substrate material 62B shown does not have the following characteristics: Figure 2A The narrower area is shown. The substrate material 62B is more planar in format.

[0099] exist Figure 3A , Figure 3B and Figure 3C The nanopore sequencing device 10B shown includes a cis electrode 30B connected to a cis trap 14B. The cis trap 14B has a lower portion including 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, which communicates with a fluid tunnel 21B to a second nanoscale opening 25B between the fluid tunnel 21B and an inverse trap 16B at the lower portion of the device 10B. As shown, the second nanoscale opening 25B is formed in a substrate material 62B. The first nanopore 18B provides a fluid channel for an electrolyte 20B to pass through between the cis trap 14B and an intermediate trap 15B. The fluid tunnel 21B provides a fluid channel for the electrolyte to pass through the second nanoscale opening 25B from the intermediate trap 15B to the inverse trap 16B. In use, an electrolyte can be filled into the cis trap 14B, the intermediate trap 15B, and the inverse trap 16B. In alternative examples, the electrolytes in cis-trap 14B, intermediate trap 15B, and trans-trap 16B can be different. In some examples, the diameter of the first nanoscale opening 23B can be equal to or smaller than the opening of the fluid tunnel 21B. The substrate for sequencing can include an array of nanopore sequencing devices. In one example of a nanopore sequencing device, the trans-trap is fluidly connected to the cis-trap via an intermediate trap and corresponding second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, there can be a common cis-trap and a common trans-trap, which are in communication with a portion or all of the nanopore sequencing devices within the array on the substrate. However, it should be understood that the array of nanopore devices can also include several cis-traps that are fluidly isolated from each other and fluidly connected to one or more corresponding trans-traps, which are fluidly isolated from each other and defined within the substrate. Multiple cis-traps may be required, for example, to enable the measurement of multiple polynucleotides on a single substrate. In some implementations, the substrate with the nanopore sequencing device array includes a common cis electrode, a common trans electrode, a common cis trap, a common trans trap, and multiple nanopore sequencing devices, such as... Figure 3AThe examples shown include each nanopore sequencing device comprising a FET sensor and a dual-well having a first nanopore and a second nanopore. Each nanopore sequencing device in a plurality of nanopore sequencing devices can individually measure resistance or signal via its associated FET sensor. In other embodiments, each nanopore sequencing device may include a multi-well and a FET sensor having three or more nanopores. 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, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of cis-wells, a plurality of trans-wells, and a plurality of nanopore sequencing devices, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode.

[0100] In one example, the cis electrode 30B and the anti electrode 34B are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis electrode and the anti electrode may be in any suitable orientation relative to each other and relative to the nanopore device. The nanopore device 10B also includes a field-effect transistor (FET) sensor 22B located between a first nanoscale opening 23B and a 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 view (… Figure 3B and Figure 3C As shown, electrolyte 20B is visible within fluid tunnel 21B and extends through channel 54B. Metal interconnects 64B and 66B are electrically connected to the source 50B and drain 52B of FET 22B via etch stop layer 38B. Metal interconnects 64B and 66B transmit data from FET sensor 22B to a control system monitoring FET sensor 22B.

[0101] exist Figure 3A In the example of the nanopore device 10B shown, a thin layer of gate oxide 56B is grown around the channel 54B; therefore, the upper surface 55B of this thin layer is fluidly exposed to the intermediate well 15B. The gate oxide 56B may have a vertical surface fluidly exposed to the electrolyte 20B in the fluid tunnel 21B. The thin layer of gate oxide separates the channel 54B from the electrolyte 20B and exposes the channel 54B of the FET sensor 22B to the electrolyte 20B. In addition to exposing the channel 54B to voltage changes through the boundary of the fluid tunnel 21B, such as… Figure 3A , Figure 3B and Figure 3C The structure shown, with its exposed upper surface 55B, significantly increases the FET's exposure to δV. MThe sensing area is increased and the LoD is improved. The thickness of the gate oxide 56B can be between about 1 nm and about 10 nm, and in some examples, the thickness is between about 2 nm and about 4 nm. The thickness of the gate oxide 56B is chosen such that, at a given potential V M In this case, a sufficiently strong electric field can induce an inversion layer of electrons or holes that form the conductive path at the boundary of the channel 54B-gate oxide 56B, so as to conduct electricity between the source 50B and the drain 52B.

[0102] 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 etchants with high anisotropy, such as fluorinated reactive ion etching, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar as some non-limiting examples.

[0103] As shown in the figure, with Figure 2A compared to, Figure 3A The trans-trap 16 in the nanopore sequencing device does not include a narrower region. In some cases, this allows for a larger trans-trap 16B. The basic operating principle remains the same for the rest of the nanopore sequencing device.

[0104] Figure 3B and Figure 3C They are respectively in Figure 3A A top-view cross-section taken on line 3-3 and line 3'-3' shows an example of a FET sensor as a nanowire transistor, namely channel 54B with a nanowire configuration.

[0105] In the nanowire transistor, 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 30B to the anti electrode 34B, and a width along a direction at least partially or substantially orthogonal to both the length and the height. In one example, the length may be at least about 10 times the width or the height. The intersection of the fluid tunnel 21B and the channel 54B in the plane defined by the length and width may be disk-shaped, such as... Figure 3B and Figure 3C As shown.

[0106] The LoD of a nanowire transistor with approximately 250 nm × 20 nm × 30 nm nanowires is approximately 3 mV, while the LoD of a nanowire transistor with approximately 10,000 nm × 100 nm × 30 nm nanowires is approximately 0.2 mV.

[0107] and Figure 3B and Figure 3C Compared to the nanowire FET sensor 22B shown, Figure 3D and Figure 3E This is a top cross-sectional view of the nanosheet FET sensor 22B'. In the nanosheet FET sensor 22B', the channel 54B' has a nanosheet configuration. A thin layer of gate oxide 56B' separates the upper 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 nm to about 10 nm, preferably about 2 nm to about 4 nm. The thickness of the gate oxide 56B' is chosen such that, at a given potential V... M In this case, a sufficiently strong electric field can induce an inversion layer of electrons or holes forming the conductive path at the boundary of the channel 54B'-gate oxide 56B', thereby enabling conduction between the source 50B' and the drain 52B'. The sensing area of ​​the FET exposed to the electrolyte 20B' is greatly increased, thereby further reducing the LoD.

[0108] 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 reverse electrode, and a width along a direction at least partially or substantially orthogonal 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 examples, the length can be at least about three, four, five, six, seven, eight, nine, ten, or more times the height, and the width can be at least about three, four, five, six, seven, eight, nine, ten, or more times the height. The intersection of the fluid tunnel 21B' and the channel 54B' in the plane defined by the length and width can be elliptical, such as... Figure 3D and Figure 3E As shown (for example, see the elliptical boundary of 56B').

[0109] Alternatively, the intersection of the fluid tunnel 21B' and the channel 54B' in the nanosheet transistor can have almost any shape and size, potentially even further increasing the sensing area of ​​the FET and thus even further reducing the LoD. Because the size and shape requirements of the fluid tunnel can be relaxed, the manufacturability of the device can be improved.

[0110] Additional examples

[0111] Figure 4A , Figure 4B and Figure 4B' It shows Figure 2A , Figure 2B and Figure 2B' Another example of a nanopore device shown is one that uses a gate-all-ring (GAA) transistor. Figure 4A This is a cross-sectional side view of the nanopore sequencing device 10C. Figure 4B Is Figure 4A A top view of the cross-section taken from line 3-3 in the image. Figure 4B' Is Figure 4A Top view of the cross section taken on line 3'-3' in the middle.

[0112] Figure 4A , Figure 4B and Figure 4B' The illustrated nanopore sequencing device 10C includes a cis electrode 30C connected to a cis trap 14C. The cis trap 14C has a lower portion including 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, which communicates with a fluid tunnel 21C to a second nanoscale opening 25C in a narrower region 17C disposed between the fluid tunnel 21C and an inverse trap 16C at the lower portion of the device 10C. As shown, the second nanoscale opening is formed in a substrate material 62C. The first nanopore 18C provides a fluid channel for an electrolyte 20C to pass between the cis trap 14C and an intermediate trap 15C. The fluid tunnel 21C provides a fluid channel for the electrolyte to pass through the second nanoscale opening 25C from the intermediate trap 15C to the inverse trap 16C. The substrate for sequencing may include an array of nanopore sequencing devices. In one example of a nanopore sequencing device, the trans trap is fluidly connected to the cis trap via an intermediate trap and corresponding second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, there may be a common cis trap and a common trans trap, which are in communication with a portion or all of the nanopore sequencing devices within the array on the substrate. However, it should be understood that the array of nanopore devices may also include a plurality of cis traps fluidly isolated from each other and fluidly connected to one or more corresponding trans traps, which are fluidly isolated from each other and defined within the substrate. Multiple cis traps may be required, for example, to enable the measurement of multiple polynucleotides on a single substrate. In some embodiments, the substrate having an array of nanopore sequencing devices includes a common cis electrode, a common trans electrode, a common cis trap, a common trans trap, and multiple nanopore sequencing devices, such as... Figure 4AThe examples shown include each nanopore sequencing device comprising a FET sensor and a dual-well having a first nanopore and a second nanopore. Each nanopore sequencing device in a plurality of nanopore sequencing devices can individually measure resistance or signal via its associated FET sensor. In other embodiments, each nanopore sequencing device may include a multi-well and a FET sensor having three or more nanopores. 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, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of cis-wells, a plurality of trans-wells, and a plurality of nanopore sequencing devices, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode.

[0113] In one example, the cis electrode 30C and the anti electrode 34C are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis electrode and the anti electrode may be in any suitable orientation relative to each other and relative to the nanopore device. The nanopore device 10C also includes a field-effect transistor (FET) sensor 22C located between a first nanoscale opening 23C and a 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 view (… Figure 4B and Figure 4B' As shown, electrolyte 20C is visible in fluid tunnel 21C and extends through channel 54C. Metal interconnects 66C and 64C are electrically connected to the source 50C and drain 52C of FET 22C via etch stop layer 38C. The metal interconnects transmit data from FET sensor 22C to the control system monitoring FET sensor 22C.

[0114] exist Figure 4AIn the nanopore sequencing device 10C shown, 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. Additionally, removing or hollowing out most of the material directly below the channel 54C also exposes the channel 54C to the electrolyte from below—this can be formed by undercutting the active region 54C of the FET sensor 22C using well-known methods. Only a thin layer of gate oxide 56C grows around the channel 54C. The upper surface 55C and lower surface 58C of the gate oxide 56C are fluidly exposed to the electrolyte 20C in the intermediate well 15C and the fluid channel 21C. The gate oxide 56C may have a vertical surface that is fluidly exposed to the electrolyte 20C in the fluid tunnel 21C. The 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 between about 1 nm and about 10 nm, and in some examples, between about 2 nm and about 4 nm. The thickness of the gate oxide 56C is chosen such that, at a given potential V... M In this case, a sufficiently strong electric field can induce an inversion layer of electrons or holes that form the conductive path at the boundary of the channel 54C-gate oxide 56C, so as to conduct electricity between the source 50C and the drain 52C.

[0115] Figure 4A The configuration of the FET sensor 22C shown allows a relatively large area of ​​the channel 54C to be exposed to the electrolyte 20C (with... Figure 2A (Compared). Therefore, channel 54C uses upper surface 55C and lower surface 58C to fluidly connect channel 54C to intermediate well 15C. Therefore, potential V M Advantageous gate controllability is achieved on channel 54C, further reducing LoD. This is in addition to exposing channel 54C to voltage variations at the gate oxide 56C at the boundary through fluid tunnel 21C, such as... Figure 4A , Figure 4B and Figure 4B' The structure of the FET sensor 22C shown, with its exposed upper surface 55C and lower surface 58C, significantly increases the FET's exposure to δV. M The sensing area was increased and the LoD was improved.

[0116] 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 etchants with high anisotropy, such as fluorinated reactive ion etching, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar as some non-limiting examples.

[0117] Membrane 24C can be either non-permeable or semi-permeable material. A first nanoscale opening 23C extends through membrane 24C. It should be understood that membrane 24C can be formed from any suitable natural or synthetic material, as described herein. In the example, membrane 24C is selected from the group consisting of lipids and biomimetic equivalents of lipids. In another example, membrane 24C is a synthetic membrane (e.g., a solid membrane, an example of which is silicon nitride), and the first nanoscale opening 23C is located in a solid nanopore extending through membrane 24C. In the example, the first nanoscale opening 23C extends through, for example: a polynucleotide nanopore disposed in the membrane; a peptide nanopore; or a solid nanopore, such as a carbon nanotube.

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

[0119] The first nanopore 18C can be any of a biological nanopore, a solid-state nanopore, a hybrid nanopore, and a synthetic nanopore. In some examples, the first nanopore 18C has two open ends and a hollow core or cavity connecting the two open ends (i.e., a first nanoscale opening 23C). When inserted into the membrane 24C, one of the open ends of the first nanopore 18C faces the cis-well 14C, and the other open end of the first nanopore 18C faces the intermediate well 15C. In some cases, the open end of the first nanopore 18C facing the intermediate well 15C is fluidly connected to the fluid tunnel 21C and may also be aligned with at least a portion of the fluid tunnel 21C. In other cases, the open end of the first nanopore 18C facing the intermediate 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 enables both fluid and electrical connections between the cis-well 14C and the intermediate well 15C. The diameter of the hollow core of the first nanopore 18C can range from about 1 nm to at most about 1 μm, and can vary along the length of the first nanopore 18C. In some examples, the opening end facing the cis-well 14C can be larger than the opening end facing the intermediate well 15C. In other examples, the opening end facing the cis-well 14C can be smaller than the opening end facing the intermediate well 15C.

[0120] Other examples

[0121] Figure 5A , Figure 5B and Figure 5B' It shows the Figure 2A , Figure 2B and Figure 2B' The modified nanoporous device shown uses a porous structure 2500D instead of... Figure 2A The second nanoscale opening 25A is shown. Figure 5A This is a side cross-sectional view of the modified exemplary device 10D. Figure 5B Is Figure 5A A top view of the cross-section taken from line 3-3 in the image. Figure 5B' Is Figure 5A Top view of the cross section taken on line 3'-3' in the middle.

[0122] Figure 5A , Figure 5B and Figure 5B' The illustrated nanopore sequencing device 10D includes a cis electrode 30D connected to a cis trap 14D. The cis trap 14D has a lower portion including a first nanopore 18D disposed within a membrane 24D. The first nanopore 18D includes a first nanoscale opening 23D defined by the first nanopore 18D, which communicates with a fluid tunnel 21D to a narrower region 17D of an inverse trap 16D at the lower portion of the device 10D. The first nanopore 18D provides a fluid channel for an electrolyte 20D to pass through between the cis trap 14D and an intermediate trap 15D. The fluid tunnel 21D provides a fluid channel for the electrolyte to pass from the intermediate trap 15D to the inverse trap 16D. A porous structure 2500D is disposed between the inverse trap 16D and the intermediate trap 15D. A substrate for sequencing may include an array of nanopore sequencing devices. In one example of a nanopore sequencing device, the inverse trap is fluidly connected to the cis trap via an intermediate trap and corresponding second and first nanoscale openings. In a substrate with a nanopore sequencing device array, there may be a common cis-well and a common trans-well, which are in communication with a portion or all of the nanopore sequencing devices within the array on the substrate. However, it should be understood that the array of nanopore devices may also include a plurality of cis-wells fluidly isolated from each other and fluidly connected to corresponding one or more trans-wells, which are fluidly isolated from each other and defined within the substrate. Multiple cis-wells may be required, for example, to enable the measurement of multiple polynucleotides on a single substrate. In some embodiments, the substrate with the nanopore sequencing device array includes a common cis-electrode, a common trans-electrode, a common cis-well, a common trans-well, and multiple nanopore sequencing devices, such as... Figure 5AThe examples shown include each nanopore sequencing device comprising a FET sensor and a dual-well having a first nanopore and a second nanopore. Each nanopore sequencing device in a plurality of nanopore sequencing devices can individually measure resistance or signal via its associated FET sensor. In other embodiments, each nanopore sequencing device may include a multi-well and a FET sensor having three or more nanopores. 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, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of cis-wells, a plurality of trans-wells, and a plurality of nanopore sequencing devices, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode.

[0123] In one example, the cis electrode 30D and the anti electrode 34D are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis electrode and the anti electrode may be in any suitable orientation relative to each other and relative to the nanopore device. The nanopore device 10D also includes a field-effect transistor (FET) sensor 22D located between a first nanoscale opening 23D and a 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 view (… Figure 5B and Figure 5B' As shown, electrolyte 20D is visible within fluid tunnel 21D and extends through channel 54D. Metal interconnects 66D and 64D are electrically connected to the source 50D and drain 52D of FET 22D via etch stop layer 38D. Metal interconnects 66D and 64D transmit data from FET sensor 22D to the control system monitoring FET sensor 22D.

[0124] exist Figure 5A In the example of the nanopore device 10D shown, a thin layer of gate oxide 56D is grown around the channel 54D; thus, the upper surface 55D of this thin layer is fluidly exposed to the intermediate well. The gate oxide 56D may have a vertical surface fluidly exposed to the electrolyte 20D in the fluid tunnel 21D. The gate oxide separates the channel 54D from the electrolyte 20D and exposes the channel 54D of the FET sensor 22D to the electrolyte 20D. The thickness of the gate oxide 56D can be between about 1 nm and about 10 nm, and in some examples, between about 2 nm and about 4 nm. The thickness of the gate oxide 56D is chosen such that, at a given potential V... M In this case, a sufficiently strong electric field can induce an inversion layer of electrons or holes that form the conductive path at the boundary of the channel 54D-gate oxide 56D, so as to conduct electricity between the source 50D and the drain 52D.

[0125] In this configuration, channel 54D has an upper surface that fluidly connects channel 54D to the electrolyte in intermediate trap 15D, such as Figure 5B As shown. By increasing the area of ​​channel 54D exposed to electrolyte 20D, the potential V M Better gate controllability is achieved on channel 54D. This is in addition to exposing channel 54D to voltage variations at the boundary of the gate oxide 56D via the fluid tunnel 21D, such as... Figure 5A , Figure 5B and Figure 5B' The structure shown, with its exposed upper surface featuring channel 54D, significantly increases the FET's exposure to δV. M The sensing area was increased and the LoD was improved.

[0126] exist Figure 2A This is part of the operation of a device, for example, defined by a second nanoscale opening 25A formed in a solid nanopore. Fabricating nanopores smaller than about 10 nm using current complementary metal-oxide-semiconductor (CMOS) technology can be challenging. However, this choice limits the voltage divider ratio D to about 0.1, which in turn reduces the change δD when the bases of the polynucleotide at the first nanoscale opening 23 change, which in turn increases the required co- and reverse bias voltages V. In some embodiments, the equivalent circuit of the nanopore device satisfies the following equation:

[0127] exist Figure 2A In device 10A, the signal detected by the FET sensor is proportional to the following equation.

[0128]

[0129] This signal is maximized when the following equation is met.

[0130]

[0131] This translates into the following requirements

[0132] R 孔 =R 蛋白质 (8)

[0133] Fabricating solid-state nanopores with similar size and resistance to protein nanopores remains challenging with current CMOS-based manufacturing techniques. Furthermore, a single solid-state nanopore meeting this requirement can exhibit varying resistance because polynucleotides (e.g., single-stranded DNA polymers) with a width of approximately 1 nm are expected to significantly alter their resistance as they pass through solid-state nanopores with openings of similar width.

[0134] On the contrary, Figure 5AIn this process, a porous structure of 2500D (e.g., a nanoporous glass frit or membrane) is used instead of the second nanoscale opening. The structure and function of the glass frit are similar to those of the glass frit used in the reference electrode. The pores in the glass frit can be randomly distributed and can form complex channels. The porosity of the glass frit is chosen such that it is sufficient to establish electrical continuity across the glass frit (i.e., large enough to allow ionic material from the electrolyte to pass through), but small enough to establish significant resistance to ionic currents. A 1mm... 2 A typical glass frit of this size has a resistivity of 1 MΩ. Therefore, a 100 nm × 100 nm glass frit can be expected to have a resistivity >1 TΩ. Typical glass frits have pore sizes of approximately a few nm and thicknesses of approximately 1 mm. Adjusting the porosity and thickness of the glass frit should allow the desired target to be achieved according to the following equation.

[0135] R 玻璃料 =R 蛋白质 (9)

[0136] Many compatible materials exist that can be used for the manufacture of glass frits. Low-κ dielectrics, such as porous low-κ dielectrics (e.g., organosilicon glasses (SiCOH), such as porous organosilicon glasses (SiCOH)), can be used and fabricated to have porosities that can be tuned up to 50%. Precursors with cyclic structures such as cyclomethylsiloxanes, such as decamethylcyclosiloxane ([(CH3)2SiO]5), are sometimes used to achieve intrinsic porosities of a few percent. Porosities up to 50% can be achieved from biphase precursors such as DMDS (dimethyl disulfide, CH3SSCH3) and α-terpinene, where the α-terpinene phase is removed by heat treatment. The structure of the resulting material can vary from worm-like mesopores arranged in a disordered manner to ordered channel-like arrays with typical pore sizes of about a few nm. Ordered porosities with periods of about tens of nm have also been demonstrated.

[0137] Additional examples

[0138] Figure 6 This is a cross-sectional, schematic, and partial cross-sectional view of yet another exemplary nanopore sequencing device 10E. Figure 6 It shows Figure 5A The modification further improves the SNR and gate controllability of the FET sensor by using a stack of channel 601.

[0139] Figure 6The illustrated nanopore sequencing device 10E includes a cis electrode 30E connected to a cis trap 14E. The cis trap 14E has a lower portion including 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, which communicates with a fluid tunnel 21E to a narrower region 17E of the trans trap 16E at the lower portion of the device 10E. The first nanopore 18E provides a fluid channel for an electrolyte 20E to pass through between the cis trap 14E and an intermediate trap 15E. The fluid tunnel 21E provides a fluid channel for the electrolyte to pass from the intermediate trap 15E to the trans trap 16E. A porous structure 2500E is disposed between the trans trap 16E and the intermediate trap 15E. A substrate for sequencing may include an array of nanopore sequencing devices. In one example of a nanopore sequencing device, the trans trap is fluidly connected to the cis trap through an intermediate trap and corresponding second and first nanoscale openings. In a substrate with a nanopore sequencing device array, there may be a common cis-well and a common trans-well, which are in communication with a portion or all of the nanopore sequencing devices within the array on the substrate. However, it should be understood that the array of nanopore devices may also include a plurality of cis-wells fluidly isolated from each other and fluidly connected to corresponding one or more trans-wells, which are fluidly isolated from each other and defined within the substrate. Multiple cis-wells may be required, for example, to enable the measurement of multiple polynucleotides on a single substrate. In some embodiments, the substrate with the nanopore sequencing device array includes a common cis-electrode, a common trans-electrode, a common cis-well, a common trans-well, and multiple nanopore sequencing devices, such as... Figure 6 The examples shown include each nanopore sequencing device comprising a FET sensor and a dual-well having a first nanopore and a second nanopore. Each nanopore sequencing device in a plurality of nanopore sequencing devices can individually measure resistance or signal via its associated FET sensor. In other embodiments, each nanopore sequencing device may include a multi-well and a FET sensor having three or more nanopores. 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, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of cis-wells, a plurality of trans-wells, and a plurality of nanopore sequencing devices, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode.

[0140] In one example, the cis electrode 30E and the anti electrode 34E are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis electrode and the anti electrode may be in any suitable orientation relative to each other and relative to the nanopore device. The nanopore device 10D also includes a field-effect transistor (FET) sensor 22E located between a first nanoscale opening 23E and a porous structure 2500E. The FET sensor includes a source (S) 50E and a drain (D) 52E. Metal interconnects 66E and 64E are electrically connected to the source 50E and drain 52E of the FET 22E via an etch stop layer 38E. The metal interconnects 66E and 64E transmit data from the FET sensor 22E to a control system that monitors the FET sensor 22E.

[0141] The FET sensor 22E is modified such that the FET also includes a stack of channels 601, which are substantially horizontally aligned and connect the source 50E to the drain 52E. In the example of the nanopore device 10E shown in Figure 5E, a thin layer of gate oxide 56E grows around the stack of channels 601. The thin layer of gate oxide separates the channel from the electrolyte 20E and exposes the channel of the FET sensor 22E to the electrolyte 20E. The thickness of the gate oxide 56E can be between about 1 nm and about 10 nm, and in some examples, between about 2 nm and about 4 nm. The thickness of the gate oxide 56E is chosen such that at a given potential V... M In this case, a sufficiently strong electric field can induce an inversion layer of electrons or holes forming the conductive path at the boundary of channel 54E-gate oxide 56E, to conduct electricity between source 50E and drain 52E. Therefore, each of the plurality of channels 605 has a gate oxide fluidly connected to the upper surface 607 and lower surface 608 of the intermediate well 15E. Each channel 605 may have a vertical surface fluidly connected to the fluid tunnel 21E. The fluid tunnel 21E extends through each of the plurality of channels. Therefore, the total FET sensing area can be increased by increasing the number of channels in the stack. By increasing the area of ​​channel 601 exposed to electrolyte 20E, the potential V... M It offers better gate controllability in the channel. This configuration significantly increases the FET's exposure to δV. M The sensing area was increased and the LoD was improved.

[0142] Figure 6 The device 10E includes a porous structure 2500E, such as a nanoporous glass frit or membrane. However, it should be recognized that this example can also use similar... Figure 2A The second nanoscale opening in the structure. However, in Figure 6In the example shown, the structure and function of the glass frit are similar to those of the glass frit used in the reference electrode. The porosity of the glass frit is chosen such that it is sufficient to establish electrical continuity across the glass frit (i.e., large enough to allow ionic material from the electrolyte to pass through), but small enough that the polymer cannot diffuse across the glass frit. The size is 1 mm. 2 The resistivity of a typical glass frit is approximately 1 MΩ, therefore a 100 nm × 100 nm glass frit can be expected to have a resistivity >1 TΩ. Typical glass frits have pore sizes of approximately a few nm and a thickness of approximately 1 mm. Adjusting the porosity and thickness of the glass frit should allow for the achievement of R... 玻璃料 The expected goal.

[0143] Other aspects and advantages of this disclosure will become apparent from the detailed description taken in conjunction with the accompanying drawings, which illustrate the principles of this disclosure by way of example.

[0144] While only certain features of the examples are shown and described herein, many modifications and variations will occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and variations.

[0145] Various modifications and variations to the described methods and compositions will be apparent to those skilled in the art without departing from the scope of the examples described herein. It should be understood that the claimed examples should not be unduly limited to the specific examples disclosed herein. In fact, various modifications that will be apparent to those skilled in the art are intended to fall within the scope of the appended claims.

[0146] Other aspects and advantages of this disclosure will become apparent from the detailed description taken in conjunction with the accompanying drawings, which illustrate the principles of this disclosure by way of example.

[0147] While only certain features have been shown and described herein, many modifications and variations will occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and variations.

[0148] Alternative examples

[0149] Figure 7A , Figure 7B and Figure 7B' It shows Figure 2A , Figure 2B and Figure 2B' Another variation of the nanopore device shown has an alternative arrangement to the fluid tunnels of a field-effect transistor. Figure 7A This is a cross-sectional side view of the nanopore sequencing device 10F. Figure 7B Is Figure 7A A top view of the cross-section taken from line 3-3 in the image. Figure 7B' Is Figure 7A Top view of the cross section taken on line 3'-3' in the middle.

[0150] Figure 7A , Figure 7B and Figure 7B' The illustrated nanopore sequencing device 10F includes a cis electrode 30F connected to a cis trap 14F. The cis trap 14F has a lower portion including 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, which communicates with a deflection fluid tunnel 21F to a second nanoscale opening 25F. The second nanoscale opening 25F is disposed in a narrow region 17F between the deflection fluid tunnel 21F and the inverse trap 16F at the lower 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 narrow region, but is more planar in layout, similar to... Figure 3A The structure shown.

[0151] The first nanopore 18F provides a fluid channel for the electrolyte 20F to pass through between the cis-well 14F and the intermediate well 15F. For example... Figure 7B As shown, the fluid tunnel 21F is positioned offset from the central portion of the device and provides a fluid channel for the electrolyte to pass through the intermediate trap 15F and reach the inverted trap 16F via the second nanoscale opening 25F.

[0152] A substrate for sequencing may include an array of nanopore sequencing devices 10F. In one example of a nanopore sequencing device, a trans trap is fluidly connected to a cis trap via an intermediate trap and corresponding second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, there may be a common cis trap and a common trans trap, which are in communication with a portion or all of the nanopore sequencing devices within the array on the substrate. However, it should be understood that the array of nanopore devices may also include a plurality of cis traps fluidly isolated from each other and fluidly connected to one or more corresponding trans traps, which are fluidly isolated from each other and defined within the substrate. Multiple cis traps may be required, for example, to enable the measurement of multiple polynucleotides on a single substrate. In some embodiments, a substrate having an array of nanopore sequencing devices includes a common cis electrode, a common trans electrode, a common cis trap, a common trans trap, and multiple nanopore sequencing devices, such as... Figure 7AThe examples shown include each nanopore sequencing device comprising a FET sensor and a dual-well having a first nanopore and a second nanopore. Each nanopore sequencing device in a plurality of nanopore sequencing devices can individually measure resistance or signal via its associated FET sensor. In other embodiments, each nanopore sequencing device may include a multi-well and a FET sensor having three or more nanopores. 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, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of cis-wells, a plurality of trans-wells, and a plurality of nanopore sequencing devices, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode.

[0153] In one example, the cis electrode 30F and the anti electrode 34F are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis electrode and the anti electrode may be in any suitable orientation relative to each other and relative to the nanopore device. The nanopore device 10F also includes a field-effect transistor (FET) sensor 22F located between a first nanoscale opening 23F and a 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... Figure 3B and Figure 3C The structure shown is illustrated. In other embodiments, the channel 54F has a nanosheet configuration, similar to... Figure 3D and Figure 3E The structure is shown. Metal interconnects 66F and 64F are electrically connected to the source 50F and drain 52F of FET 22F via an etched stop layer 38F. The metal interconnects transmit data from FET sensor 22F to a control system that monitors FET sensor 22F. In an alternative embodiment, nanopore sequencing device 10F can use a porous structure instead of the second nanoscale opening 25F, similar to... Figure 5A The structure shown.

[0154] As shown in the top view of the cross section ( Figure 7B and Figure 7B' As shown in the diagram, fluid tunnel 21F is offset from channel 54F. In other words, fluid tunnel 21F does not extend through channel 54F, and therefore... Figure 7A Not visible in the cross-sectional side view. Instead, the fluid tunnel 21F extends through the interlayer dielectric 68F surrounding the channel 54F. Figure 7B and Figure 7B' Electrolyte 20F can be seen within fluid tunnel 21F. The boundary of fluid tunnel 21F can be circular, such as... Figure 7B and Figure 7B' As shown. In other embodiments, the boundary of the fluid tunnel 21F can be elliptical, such as... Figure 3D and Figure 3E As shown. Alternatively, the boundaries of the fluid tunnel 21F can have virtually any shape and size. In some embodiments, the FET sensor 22F may include a channel stack, similar to... Figure 6 The structure shown is such that the fluid tunnels do not extend through the channel stacks.

[0155] Offset fluid tunnel 21F relative to Figure 7A , Figure 7B and Figure 7B' One non-limiting benefit of the illustrated channel 54F arrangement is a simpler manufacturing process. Etching holes / openings in the channel can interfere with the device's gate oxide and require additional oxide regrowth steps. Figure 7A , Figure 7B and Figure 7B' The illustrated implementation can avoid etching holes or openings in the source-drain channel.

[0156] 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 etchants with high anisotropy, such as fluorinated reactive ion etching, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar as some non-limiting examples.

[0157] In one example, the source, drain, and channel of the FET sensor 22F can be formed of silicon, and the surface of the silicon can be thermally oxidized to form a gate oxide on the channel of the FET sensor 22F.

[0158] exist Figure 7A In the nanopore sequencing device 10F shown, most of the material directly above line 3-3, which separates channel 54F from electrolyte 20F, is removed, exposing channel 54F of the FET sensor 22F to electrolyte 20F. Figure 7A As shown, a portion of channel 54F is exposed to the electrolyte from below. In other embodiments, similar to Figure 4AThe structure shown allows for the removal or hollowing out of most of the material directly beneath the channel 54F, exposing a larger portion of the channel 54F to the electrolyte from below—this can be formed by undercutting the active region 54F of the FET sensor 22F using well-known methods. Only a thin layer of gate oxide 56F grows around the channel 54F. The upper surface 55F and lower surface 58F of the gate oxide 56F are fluidly exposed to the electrolyte 20F in the intermediate well 15F. The 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 between about 1 nm and about 10 nm, and in some examples, between about 2 nm and about 4 nm. The thickness of the gate oxide 56F is chosen such that at a given potential V... M In this case, a sufficiently strong electric field can induce an inversion layer of electrons or holes that form the conductive path at the boundary of the channel 54F-gate oxide 56F, so as to conduct electricity between the source 50F and the drain 52F.

[0159] Membrane 24F can be either non-permeable or semi-permeable material. A first nanoscale opening 23F extends through membrane 24F. It should be understood that membrane 24F can be formed from any suitable natural or synthetic material, as described herein. In the examples, membrane 24F is selected from the group consisting of lipids and biomimetic equivalents of lipids. In another example, membrane 24F is a synthetic membrane (e.g., a solid membrane, an example of which is silicon nitride), and the first nanoscale opening 23F is located in a solid nanopore extending through membrane 24F. In the examples, the first nanoscale opening 23F extends through, for example: a polynucleotide nanopore disposed in the membrane; a peptide nanopore; or a solid nanopore, such as a carbon nanotube.

[0160] The first nanopore 18F can be any of a biological nanopore, a solid-state nanopore, a hybrid nanopore, and a synthetic nanopore. In some examples, the first nanopore 18F has two open ends and a hollow core or cavity connecting the two open ends (i.e., a first nanoscale opening 23F). When inserted into the membrane 24F, one of the open ends of the first nanopore 18F faces the cis-well 14F, and the other open end of the first nanopore 18F faces the intermediate well 15F. In some cases, the open end of the first nanopore 18F facing the intermediate well 15F is fluidly connected to the fluid tunnel 21F and may also be aligned with at least a portion of the offset fluid tunnel 21F. In other cases, the open end of the first nanopore 18F facing the intermediate well 15F is fluidly connected to the fluid tunnel 21F but not aligned with the offset fluid tunnel 21F. The hollow core of the first nanopore 18F enables both fluid and electrical connections between the cis-well 14F and the intermediate well 15F. The diameter of the hollow core of the first nanopore 18F can range from about 1 nm to at most about 1 μm, and can vary along the length of the first nanopore 18F. In some examples, the opening end facing the cis-well 14F can be larger than the opening end facing the intermediate well 15F. In other examples, the opening end facing the cis-well 14F can be smaller than the opening end facing the intermediate well 15F.

[0161] Methods of using the nanopore sequencing device 10F may include introducing an electrolyte 20F into each of the cis-trap 14F, trans-trap 16F, intermediate trap 15F, and fluid tunnel 21F. After introducing the electrolyte, the method may include providing the polynucleotide to be sequenced into the cis-trap 14F. After providing the polynucleotide, the method may include applying a bias voltage between the cis-electrode 30F and the trans-electrode 34F. The bias voltage drives the polynucleotide from the cis-trap 14F to the intermediate trap 15F through a first nanoscale opening 23F. As the polynucleotide passes through the first nanoscale opening 23F, the resistance of the first nanoscale opening changes in response to the base identity in the polynucleotide at the first nanoscale opening. As a result, the potential (V) of the electrolyte 20F in the intermediate trap 15F (or equivalently, the offset fluid tunnel 21F) changes. M The potential (V) varies depending on the base identity. M In reality, this is the gate voltage applied to the FET, which regulates the conductivity of the channel 54F. Therefore, a measurement of the FET's response can determine the identity of the bases.

[0162] Figure 8 Another variant of the nanopore device is shown, which utilizes a vertical field-effect transistor, allowing the source-drain channel to be oriented vertically along one side of the fluid path through the device instead of being etched to form a fluid tunnel, as explained below. Figure 8 This is a cross-sectional side view of the 810G vertical FET nanopore sequencing device.

[0163] Figure 8 The illustrated nanopore sequencing device 810G includes a cis electrode 830G connected to a cis trap 814G. The cis trap 814G has a lower portion including 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, which is in fluid communication with a second nanoscale opening 825G. The second nanoscale opening 825G may be disposed in a narrower region 817G of an inverse trap 816G at the lower 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 narrower region, but is more planar in layout, similar to... Figure 3A The structure is shown. A first nanopore 818G provides a fluid channel for electrolyte 820G to pass through between the cis-well 814G and the intermediate well 815G. A substrate for sequencing may include an array of nanopore sequencing devices. In one example of a nanopore sequencing device, the trans-well is fluidly connected to the cis-well via an intermediate well and corresponding second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, there may be a common cis-well and a common trans-well, which are in communication with a portion or all of the nanopore sequencing devices within the array on the substrate. However, it should be understood that the array of nanopore devices may also include a plurality of cis-wells fluidly isolated from each other and fluidly connected to one or more corresponding trans-wells, which are fluidly isolated from each other and defined within the substrate. Multiple cis-wells may be required, for example, to enable the measurement of multiple polynucleotides on a single substrate. In some embodiments, a substrate having an array of nanopore sequencing devices includes a common cis-electrode, a common trans-electrode, a common cis-well, a common trans-well, and multiple nanopore sequencing devices, such as... Figure 8 The examples shown include each nanopore sequencing device comprising a FET sensor and a dual-well having a first nanopore and a second nanopore. Each nanopore sequencing device in a plurality of nanopore sequencing devices can individually measure resistance or signal via its associated FET sensor. In other embodiments, each nanopore sequencing device may include a multi-well and a FET sensor having three or more nanopores. 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, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of cis-wells, a plurality of trans-wells, and a plurality of nanopore sequencing devices, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode.

[0164] In one example, the cis electrode 830G and the anti 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 anti electrode can be in any suitable orientation relative to each other and relative to the nanopore device. The nanopore device 810G also includes a vertical field-effect transistor (FET) sensor located between a first nanoscale opening 823G and a 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 the direction from the cis electrode 830G to the anti electrode 834G. In some embodiments, the channel has a nanowire configuration, similar to... Figure 3B and Figure 3C The structure is shown. In other embodiments, the channel has a nanosheet configuration, similar to... Figure 3D and Figure 3E The structure is shown. Metal interconnects 866G and 864G are electrically connected to the source 850G and drain 852G of the FET. The metal interconnects transmit data from the FET sensor to the control system that monitors the FET sensor. In an alternative embodiment, the nanopore sequencing device 810G can use a porous structure instead of the second nanoscale opening 825G, similar to... Figure 5A The structure shown.

[0165] like Figure 8 As shown, the source 850G, channel, and drain 852G of a vertical FET sensor are vertically stacked. The vertical FET is disposed on the lateral side of the intermediate well 815G. In one example, 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 surface of the gate oxide 856G is fluidly exposed to the electrolyte 820G in the intermediate well 815G. A 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 between about 1 nm and about 10 nm, and in some examples, between about 2 nm and about 4 nm. The thickness of the gate oxide 856G is chosen such that at a given potential V M In such cases, a sufficiently strong electric field can induce an inversion layer of electrons or holes that forms the conductive path at the channel-gate oxide boundary, thereby enabling conduction between the source 850G and the drain 852G. In some embodiments, the FET sensor may include multiple vertical source-drain channels arranged in parallel along the lateral side of the intermediate well.

[0166] One non-limiting benefit of vertical FET sensors is that it may be unnecessary to etch fluid tunnels through the FET channel. Etching holes / openings in the channel can interfere with the device's gate oxide and require additional oxide regrowth steps. Figure 8 The embodiment shown, which has a vertical FET arranged on the lateral side of the intermediate well 815G, can avoid etching holes or openings in the source-drain channel.

[0167] 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 etchants with high anisotropy, such as fluorinated reactive ion etching, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar as some non-limiting examples.

[0168] Membrane 824G can be either non-permeable or semi-permeable material. A first nanoscale opening 823G extends through membrane 824G. It should be understood that membrane 824G can be formed from any suitable natural or synthetic material, as described herein. In the example, membrane 824G is selected from the group consisting of lipids and biomimetic equivalents of lipids. In another example, membrane 824G is a synthetic membrane (e.g., a solid membrane, an example of which is silicon nitride), and the first nanoscale opening 823G is located in a solid nanopore extending through membrane 824G. In the example, the first nanoscale opening 823G extends through, for example: a polynucleotide nanopore disposed in the membrane; a peptide nanopore; or a solid nanopore, such as a carbon nanotube.

[0169] The first nanopore 818G can be any of a biological nanopore, a solid-state nanopore, a hybrid nanopore, or a synthetic nanopore. In some examples, the first nanopore 818G has two open ends and a hollow core or cavity connecting the two open ends (i.e., a first nanoscale opening 823G). When inserted into the membrane 824G, one of the open ends of the first nanopore 818G faces the cis-well 814G, and the other open end of the first nanopore 818G faces the intermediate well 815G. The hollow core of the first nanopore 818G enables fluid and electrical connections between the cis-well 814G and the intermediate well 815G. The diameter of the hollow core of the first nanopore 818G can range from about 1 nm to at most about 1 μm and can vary along the length of the first nanopore 818G. In some examples, the open end facing the cis-well 814G can be larger than the open end facing the intermediate well 815G. In other examples, the opening facing the cis-well 814G may be smaller than the opening facing the intermediate well 815G.

[0170] Methods of using the nanopore sequencing device 810G may include introducing an electrolyte 820G into each of the cis-trap 814G, the trans-trap 816G, and the intermediate trap 815G. After introducing the electrolyte, the method may include providing the polynucleotide to be sequenced into the cis-trap 814G. After providing the polynucleotide, the method may include applying a bias voltage between the cis-electrode 830G and the trans-electrode 834G. The bias voltage drives the polynucleotide from the cis-trap 814G to the intermediate trap 815G through a first nanoscale opening 823G. As the polynucleotide passes through the first nanoscale opening 823G, the resistance of the first nanoscale opening changes in response to the base identity in the polynucleotide at the first nanoscale opening. As a result, the potential (V) of the electrolyte 820G in the intermediate trap 815G... M The potential (V) varies depending on the base identity. M In reality, this is the gate voltage applied to the FET, which regulates the conductivity of the FET channel. Therefore, a measurement of the FET's response can determine the identity of the bases.

[0171] Figure 9 Another variation of a nanopore sequencing device with a field-effect transistor (FET) having a non-Faraday metal electrode is shown. In this embodiment, the FET has a non-Faraday metal electrode comprising a metallic structure that does not participate in the Faraday processes in the nanopore sequencing device; that is, no electrochemical reaction occurs at the metallic structure. The non-Faraday metal electrode is used to detect the potential of the electrolyte in the intermediate trap and transmits this potential as a detected signal to the FET. This design means that the FET can detect the potential of the electrolyte without being exposed to it. Figure 9 This is a cross-sectional side view of the 910H nanopore sequencing device.

[0172] Figure 9 The illustrated nanopore sequencing device 910H includes a cis electrode 930H connected to a cis trap 914H. The cis trap 914H has a lower portion including 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, which is in fluid communication with a second nanoscale opening 925H. The second nanoscale opening 925H may be disposed in a narrower region 917H of an inverse trap 916H at the lower 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 narrower region, but is more planar in layout, similar to... Figure 3A The structure is shown. The first nanopore 918H provides a fluid channel for the electrolyte 920H to pass through between the cis-trap 914H and the intermediate trap 915H.

[0173] In one example, the cis electrode 930H and the anti electrode 934H are at least substantially parallel to each other in at least a substantially horizontal direction. In other examples, the cis electrode and the anti electrode can be in any suitable orientation relative to each other and relative to the nanopore device. The nanopore device 910H also includes a field-effect transistor (FET) sensor 922H located between a first nanoscale opening 923H and a 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 horizontal. In some embodiments, the FET channel has a nanowire configuration, similar to... Figure 3B and Figure 3C The structure is shown. In other embodiments, the FET channel has a nanosheet configuration, similar to... Figure 3D and Figure 3E The structure is shown. Metal interconnects 966H and 964H are electrically connected to the source 950H and drain 952H of the FET. Metal interconnects 966H and 964H transmit data from the FET sensor to a control system (not shown) that monitors the FET sensor. In an alternative embodiment, the nanopore sequencing device 910H can use a porous structure instead of the second nanoscale opening 925H, similar to... Figure 5A The structure shown.

[0174] like Figure 9 As shown, the FET sensor 922H does not directly contact the electrolyte. In one example, 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 956H on the channel of the FET sensor. Figure 9 As shown, the gate oxide 956H is non-fluidically exposed to the electrolyte 920H in the intermediate well 915H. Conversely, the non-Radidatic metal electrode structure 999H is exposed to the electrolyte. The metal structure 999H is used to detect the potential of the electrolyte in the intermediate well and transmit the detected signal to the FET. Compared to the size of the intermediate well (approximately a few μm), the path length or feature size of the metal structure 999H can be approximately 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 in between.

[0175] The size and shape of the metal structure 999H can be appropriately chosen to avoid high parasitic capacitance in the system. The metal structure 999H can be made of a non-Radiladian resistant metal relative to the electrolyte. The metal structure 999H can be made of platinum, iridium, ruthenium, palladium, tantalum, gold, or any combination thereof. No electrochemical reaction occurs at the metal structure. In some embodiments, the metal structure 999H can 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, wherein the fins may be arranged partially vertically or horizontally. Using the metal structure 999H to contact the electrolyte allows the FET to be decoupled from the intermediate well, which may be easier to manufacture in some embodiments. Decoupling the size of the FET from the size of the intermediate well also allows for a larger FET, which can allow for higher signal detection sensitivity and lower noise levels. This configuration also allows the size of the FET (which determines the limits of signal detection) to be decoupled from the size of the metal structure 999H, and thus provides greater design flexibility.

[0176] The thickness of the gate oxide 956H can be between about 1 nm and about 10 nm, and in some examples, between about 2 nm and about 4 nm. The thickness of the gate oxide 956H is chosen such that the potential V in a given intermediate well... M In this case, a sufficiently strong electric field can induce an inversion layer of electrons or holes that form the conductive path at the channel-gate oxide boundary, enabling conduction 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 etchants with high anisotropy, such as fluorinated reactive ion etching, including CHF3 / O2, C2F6, C3F8, and C5F8 / CO / O2 / Ar as some non-limiting examples.

[0177] The membrane 924H can be any of a non-permeable or semi-permeable material. A 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 examples, the membrane 924H is selected from the group consisting of lipids and biomimetic equivalents of lipids. In another example, the membrane 924H is a synthetic membrane (e.g., a solid membrane, an example of which is silicon nitride), and the first nanoscale opening 923H is located in a solid nanopore extending through the membrane 924H. In examples, the first nanoscale opening 923H extends through, for example: a polynucleotide nanopore disposed in the membrane; a peptide nanopore; or a solid nanopore, for example, a carbon nanotube. The first nanopore 918H can be any of a biological nanopore, a solid nanopore, a hybrid nanopore, and a synthetic nanopore. In some examples, the first nanopore 918H has two open ends and a hollow core or hole connecting the two open ends (i.e., the first nanoscale opening 923H). When inserted into membrane 924H, one of the opening ends of the first nanopore 918H faces the cis-well 914H, and the other opening end of the first nanopore 918H faces the intermediate well 915H. The hollow core of the first nanopore 918H enables fluid and electrical connections between the cis-well 914H and the intermediate well 915H. The diameter of the hollow core of the first nanopore 918H can range from about 1 nm to at most about 1 μm and can vary along the length of the first nanopore 918H. In some examples, the opening end facing the cis-well 914H can be larger than the opening end facing the intermediate well 915H. In other examples, the opening end facing the cis-well 914H can be smaller than the opening end facing the intermediate well 915H.

[0178] Methods using the nanopore sequencing device 910H may include introducing an electrolyte 920H into each of the cis-trap 914H, trans-trap 916H, and intermediate trap 915H. After introducing the electrolyte, the method may include providing the polynucleotide to be sequenced into the cis-trap 914H. After providing the polynucleotide, the method may include applying a bias voltage between the cis-electrode 930H and the trans-electrode 934H. In some embodiments, the bias voltage may drive the polynucleotide from the cis-trap 914H to the intermediate trap 915H through a first nanoscale opening 923H. As the polynucleotide passes through the first nanoscale opening 923H, the resistance of the first nanoscale opening changes in response to the base identity of the polynucleotide at the first nanoscale opening. In alternative embodiments, the polynucleotide does not pass through the first nanoscale opening, but a tag or label of the nucleotide incorporated by a polymerase acting on the polynucleotide may pass through the first nanoscale opening or may temporarily reside in the first nanoscale opening. Therefore, the resistance of the first nanoscale opening changes in response to the identity of the incorporated nucleotide, which is complementary to the base identity of the polynucleotide. As a result, the potential (V) of the electrolyte 920H in the intermediate trap 915H is...M The potential (V) varies depending on the base identity within the polynucleotide. M In reality, this is the gate voltage applied to the FET, which regulates the conductivity of the FET channel. Therefore, measurements of the FET's response can determine the identity of the bases in the polynucleotide.

[0179] The substrate used for sequencing can include an array of nanopore sequencing devices, such as... Figure 9 As shown in the examples. In one example of a nanopore sequencing device, the trans trap is fluidly connected to the cis trap via an intermediate trap and corresponding second and first nanoscale openings. In a substrate having an array of nanopore sequencing devices, there may be a common cis trap and a common trans trap, which are in communication with a portion or all of the nanopore sequencing devices within the array on the substrate. However, it should be understood that the array of nanopore devices may also include a plurality of cis traps fluidly isolated from each other and fluidly connected to one or more corresponding trans traps, which are fluidly isolated from each other and defined in the substrate. Multiple cis traps may be required, for example, to enable the measurement of multiple polynucleotides on a single substrate. In some embodiments, a substrate having an array of nanopore sequencing devices includes a common cis electrode, a common trans electrode, a common cis trap, a common trans trap, and multiple nanopore sequencing devices, such as Figure 9 The examples shown include each nanopore sequencing device comprising a FET sensor and a dual-well having a first nanopore and a second nanopore. Each nanopore sequencing device in a plurality of nanopore sequencing devices can individually measure resistance or signal via its associated FET sensor. In other embodiments, each nanopore sequencing device may include a multi-well and a FET sensor having three or more nanopores. 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, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode. In other embodiments, a substrate having an array of nanopore sequencing devices comprises a plurality of cis-wells, a plurality of trans-wells, and a plurality of nanopore sequencing devices, wherein each nanopore sequencing device can be individually addressed with an individual trans electrode.

[0180] definition

[0181] All technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0182] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, references to “a sequence” can include multiple such sequences, and so on.

[0183] The terms include, encompass, contain, and various forms of these terms are synonymous with each other and are intended to be equally broad. Furthermore, unless explicitly stated otherwise, examples of including or having one or more elements with a particular attribute may include additional elements, whether or not the additional elements have that attribute.

[0184] As used herein, the terms "fluidly connected," "fluidly connected," and "fluidly coupled" refer to two spatial regions connected together so that a liquid or gas can flow between the two spatial regions. For example, a cis-trap can be fluidly connected to an inverse trap via an intermediate trap, a fluid tunnel, and a narrower region, allowing at least a portion of the electrolyte to flow between the connected traps. The two spatial regions can be fluidly connected via first and second nanoscale openings or via one or more valves, flow restrictors, or other fluid components used to control or regulate the flow of fluid through the system.

[0185] As used herein, the term "gap region" refers to a region in a substrate / solid support or membrane, or on a surface, that separates from other regions, areas, or features associated with the support, membrane, or surface. For example, a gap region in a membrane can separate one nanopore of an array from another nanopore of the array. As another example, a gap region in a substrate can separate one inverted well from another inverted well. The two separated regions can be discrete, i.e., lacking physical contact with each other. In many examples, the gap region is continuous while the region is discrete, for example, for multiple nanopores defined in a membrane that is continuous in other respects, or for multiple wells defined in a substrate / support that is continuous in other respects. The separation provided by the gap region can be partial or complete. The gap region can have a different surface material than the surface material of the feature defined in the surface. For example, the surface material at the gap region can be a lipid material, and the nanopores formed in the lipid material can have an amount or concentration of peptides exceeding that present at the gap region. In some examples, the peptide may not be present at the gap region.

[0186] As used herein, the term "membrane" refers to a non-permeable or semi-permeable barrier or other sheet separating two liquid / gel chambers (e.g., a cis-trap and a fluid cavity), which may contain the same or different compositions. The permeability of a membrane to any given substance depends on the properties of the membrane. In some examples, the membrane may be ionicly, electrically, and / or fluidically impermeable. For example, a lipid membrane may be ionicly impermeable (i.e., not allowing any ion transport through), but may be at least partially permeable to water (e.g., with a water diffusivity in the range of about 40 μm / s to about 100 μm / s). For another example, a synthetic / solid membrane (an example of which is silicon nitride) may be ionicly, electrically, and fluidically impermeable (i.e., diffusion of all these substances is zero). Any membrane may be used according to this disclosure, provided that the membrane may include transmembrane nanoscale openings and can maintain a potential difference across the membrane. The membrane may be a monolayer or a multilayer membrane. A multilayer membrane comprises two or more layers, each of which is a non-permeable or semi-permeable material.

[0187] Membranes can be formed from materials of biological or non-biological origin. Biologically derived materials are those derived from or isolated from the biological environment (such as organisms or cells), or those that are synthetically manufactured from biologically available structures (e.g., biomimetic materials).

[0188] Exemplary membranes made from biologically derived materials include a monolayer formed of bolalipids. Another exemplary membrane made from biologically derived materials includes a lipid bilayer. Suitable lipid bilayers include, for example, cell membranes, organelle membranes, liposomes, planar lipid bilayers, and supported lipid bilayers. A lipid bilayer can be formed, for example, from two opposing phospholipid layers arranged such that their hydrophobic tail groups face each other to form a hydrophobic interior, while the hydrophilic head groups of the lipids face outward toward the aqueous environment on each side of the bilayer. A lipid bilayer can also be formed, for example, by a method in which a lipid monolayer is carried at an aqueous / air interface through either side of a pore substantially perpendicular to that interface. Typically, the lipid is added to the surface of an aqueous electrolyte solution by first dissolving the lipid in an organic solvent and then evaporating a drop of solvent onto the aqueous surface on either side of the pore. Once the organic solvent has at least partially evaporated, the solution / air interface on either side of the pore physically moves up and down through the pore until a bilayer is formed. Other suitable methods for bilayer formation include tip impregnation, coating bilayers, and patch clamps for liposome bilayers. Any other method for obtaining or generating lipid bilayers may also be used.

[0189] Non-biological materials can also be used as membranes. Some of these materials are solid and can form solid membranes, while others can form thin liquid films or membranes. Solid membranes can be monolayers, such as coatings or films on a supporting substrate (i.e., solid supports), or stand-alone elements. Solid membranes can also be composites of multilayer materials with a sandwich configuration. Any non-biological material can be used, as long as the resulting membrane can include transmembrane nanoscale openings and maintain a potential difference across the membrane. Membranes can include organic materials, inorganic materials, or both. Examples of suitable solid materials include, for example, microelectronic materials, insulating materials (e.g., silicon nitride (Si3N4), alumina (Al2O3), hafnium oxide (HfO2), tantalum pentoxide (Ta2O5), silicon oxide (SiO2), etc.), some organic and inorganic polymers (e.g., polyamides, plastics such as polytetrafluoroethylene (PTFE), or elastomers such as two-component addition-cured silicone rubber), and glass. Furthermore, solid-state films can be made from monolayer graphene (which is a densely packed, atomically thin sheet of carbon atoms arranged in a two-dimensional honeycomb lattice), multilayer graphene, or one or more layers of graphene mixed with one or more other solid materials. Graphene-containing solid-state films may include at least one graphene layer, which is a graphene nanoribbon or graphene nanogap, and can be used as an electrosensor to characterize target polynucleotides. It should be understood that solid-state films can be fabricated by any suitable method, such as chemical vapor deposition (CVD). In one example, a graphene film can be fabricated by CVD or by exfoliation from graphite. Examples of suitable thin liquid film materials that can be used include diblock copolymers or triblock copolymers, such as the amphiphilic PMOXA-PDMS-PMOXAABA triblock copolymer.

[0190] As used herein, the term "nanopore" is intended to refer to a hollow structure discretely contained within or defined in a membrane and extending through the membrane, which allows ions, currents, and / or fluids to pass from one side of the membrane to the other. For example, a membrane that inhibits the passage of ions or water-soluble molecules may include a nanopore structure that extends through the membrane to allow ions or water-soluble molecules to pass from one side of the membrane (through nanoscale openings extending through the nanopore structure) to the other side. The diameter of the nanoscale openings extending through the nanopore structure may vary along its length (i.e., from one side of the membrane to the other), but is at the nanoscale at any point (i.e., from about 1 nm to about 100 nm, or to less than 1000 nm). Examples of nanopores include, for example, biological nanopores, solid-state nanopores, and hybrid biological and solid-state nanopores.

[0191] As used herein, the term "diameter" is intended to represent the longest straight line that can be inscribed in the cross-section of a nanoscale opening, through which the centroid of the cross-section of the nanoscale opening lies. It should be understood that the nanoscale opening may or may not have a circular or substantially circular cross-section (the cross-section of the nanoscale opening is substantially parallel to the cis / trans electrodes). Furthermore, the cross-section may be regular or irregular in shape.

[0192] As used herein, the term "bio-nanopore" is intended to refer to nanopores whose structural portions are made of biologically derived materials. Biologically derived means materials derived from or isolated from the biological environment (such as organisms or cells), or synthetically manufactured forms of biologically available structures. Bio-nanopores include, for example, peptide nanopores and polynucleotide nanopores.

[0193] As used herein, the term "peptide nanopore" is intended to refer to a protein / peptide that extends across a membrane and allows ions, electric currents, polymers (such as DNA or peptides), or other molecules and / or fluids of suitable size and charge to flow from one side of the membrane to the other. Peptide nanopores can be monomers, homopolymers, or hybrids. Structures of peptide nanopores include, for example, α-helical bundle nanopores and β-barrel nanopores. Exemplary peptide nanopores include α-hemolysin, Mycobacterium smegmatis porin A (MspA), bacitracin A, maltose porin, OmpF, OmpC, PhoE, Tsx, F-pillaries, etc. The protein α-hemolysin is naturally present in cell membranes, where it acts as a pore for the transport of ions or molecules into and out of the cell. Mycobacterium smegmatis porin A (MspA) is a membrane porin produced by mycobacteria that allows hydrophilic molecules to enter the bacteria. MspA forms tightly interconnected octamers and transmembrane β-barrels, which resemble goblet cups and contain a central pore.

[0194] Peptide nanopores can be synthetic. Synthetic peptide nanopores include protein-like amino acid sequences that do not exist in nature. Protein-like amino acid sequences can include some amino acids that are known to exist but do not form the basis of proteins (i.e., non-proteinogenic amino acids). Protein-like amino acid sequences can be synthesized artificially rather than expressed in organisms and then purified / isolated.

[0195] As used herein, the term "polynucleotide nanopore" is intended to encompass polynucleotides that extend across a membrane and allow ions, electric currents, and / or fluids to flow from one side of the membrane to the other. Polynucleotide pores can include, for example, polynucleotide origami (e.g., nanoscale folding of DNA to create nanopores).

[0196] As used herein, the term "solid-state nanopore" is intended to refer to nanopores whose structural portion is defined by a solid membrane and includes materials of non-biological origin (i.e., not biologically derived). Solid-state nanopores can be formed from inorganic or organic materials. Solid-state nanopores include, for example, silicon nitride nanopores, silica nanopores, and graphene nanopores.

[0197] The nanopores disclosed herein can be hybrid nanopores. A “hybrid nanopore” refers to a nanopore that incorporates materials of both biological and non-biological origin. Examples of hybrid nanopores include peptide-solid hybrid nanopores and polynucleotide-solid nanopores.

[0198] As used herein, the term "nanopore sequencer" refers to any device disclosed herein that can be used for nanopore sequencing. In the examples disclosed herein, during nanopore sequencing, a nanopore is immersed in an electrolyte disclosed herein, and a potential difference is applied across the membrane. In the examples, the potential difference is an electrical potential difference or an electrochemical potential difference. A potential difference can be applied across the membrane by a voltage source that injects or applies a current to at least one ion of the electrolyte contained in the cis-well or one or more trans-wells. An electrochemical potential difference can be established by a combination of the difference in the ionic composition of the cis- and trans-wells and the potential. The different ionic compositions can be, for example, different ions in each well or different concentrations of the same ion in each well.

[0199] Applying a potential difference across the nanopore can force nucleic acid translocation through the first nanoscale opening 23 (e.g., in...). Figure 2A (Shown and described in more detail below). One or more signals corresponding to the translocation of nucleotides through the nanopore are generated. Thus, when a target polynucleotide or mononucleotide, or a probe derived from a target polynucleotide or mononucleotide, passes through the nanopore, the transmembrane current changes due to base-dependent (or probe-dependent) blockage, such as at the contraction site. The signal from this current change can be measured using any of a variety of methods. Each signal is unique for the type of nucleotide (or probe) in the nanopore, making the resulting signal usable for characterizing the polynucleotide. For example, the identity of one or more types of nucleotides (or probes) that generate characteristic signals can be determined.

[0200] As used herein, “nucleotide” includes a nitrogenous 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. The nitrogenous heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G) and their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U) and their modified derivatives or analogs. The C-1 atom of the deoxyribose is bonded to the N-1 of the pyrimidine or the N-9 of the purine. The phosphate group can be in the form of a monophosphate, diphosphate, or triphosphate. These nucleotides are natural nucleotides, but it should be further understood that non-natural nucleotides, modified nucleotides, or analogs of the aforementioned nucleotides may also be used.

[0201] As used herein, the term "signal" is intended to mean an indicator that represents information. Signals include, for example, electrical signals and optical signals. The term "electrical signal" refers to an indicator of the electrical quality that represents information. Indicators can be, for example, current, voltage, tunneling, resistance, potential, conductance, or transverse electrical effects. "Electronic current" or "current" refers to the flow of charge. In the example, an electrical signal could be a current passing through a nanopore, and the current could flow when a potential difference is applied across the nanopore.

[0202] The term "substrate" refers to a rigid solid support that is insoluble in aqueous liquids and cannot allow liquid to pass through without pores, ports, or other similar liquid conduits. In the examples disclosed herein, the substrate may have a trap or chamber defined therein. Examples of suitable substrates include glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutene, polyurethane, polytetrafluoroethylene (PTFE) (such as those from Chemours) ), cyclic olefin / cyclic olefin polymers (COP) (such as those from Zeon) (e.g., polyimide), nylon, ceramics, silica or silica-based materials, silicon and modified silicon, carbon, metals, inorganic glass and fiber bundles.

[0203] The terms top, bottom, lower, upper, and above are used herein to describe various components of the device / nanopore sequencer and / or apparatus. It should be understood that these directional terms are not intended to suggest a particular orientation, but rather to specify the relative orientation between components. The use of directional terms should not be construed as limiting the examples disclosed herein to any particular orientation. As used herein, the terms “upper,” “lower,” “vertical,” “horizontal,” etc., indicate a relative orientation.

[0204] As used herein, the terms “trap,” “cavity,” and “chamber” are used synonymously and refer to a discrete feature defined in a device that may contain a fluid (e.g., liquid, gel, gas). A cis-trap is a fluid system comprising a cis-electrode or a chamber partially defined by a cis-electrode and fluidly connected to a FET, which in turn is fluidly connected to an inverse trap / chamber. Examples of arrays of devices of the present invention may have one or more cis-traps. An inverse trap is a single chamber comprising its own inverse electrode or a chamber partially defined by that inverse electrode and fluidly connected to a cis-trap. In examples comprising multiple inverse traps, each inverse trap is electrically isolated from each other. Furthermore, it should be understood that the cross-section of a trap, taken from a surface at least partially defining the trap parallel to the substrate, can be curved, square, polygonal, hyperbolic, conical, angular, etc.

[0205] As used herein, a "field-effect transistor" or "FET" typically includes doped source / drain regions formed from a semiconductor material (e.g., silicon, germanium, gallium arsenide, silicon carbide, etc.) and 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 contain a different material than those of a p-FET device. In some examples, the source / drain regions or channels may be undoped. Doped regions can be formed by adding dopant atoms to the intrinsic semiconductor. This alters the electron and hole carrier concentrations of the intrinsic semiconductor at thermal equilibrium. Doped regions can be p-type or n-type. As used herein, "p-type" refers to the addition of an impurity to the intrinsic semiconductor, resulting in valence electron defects. 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 the intrinsic semiconductor. For silicon, exemplary n-type dopants (i.e., impurities) include, but are not limited to, antimony, arsenic, and phosphorus. Dopants can be introduced by ion implantation or plasma doping.

[0206] For example, in an integrated circuit having multiple metal-oxide-semiconductor field-effect transistors (MOSFETs), each MOSFET has a source and a drain formed in an active region of a semiconductor layer by implanting n-type or p-type impurities into the semiconductor material layer. Between the source and drain is a channel (or body) region. Above the body region is the 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 from the source to the drain through the channel region. A current is induced in the channel region by a voltage applied to the gate electrode.

[0207] Non-planar transistor device architectures, such as nanosheet (or nanowire) transistors, can provide increased device density and improved performance compared to planar transistors. A “gate-all-around” transistor is a transistor in which the gate is configured to surround a channel. A “nanosheet transistor” refers to a type of FET that can include multiple stacked nanosheets extending between a pair of source / drain regions to form a channel. Compared to conventional planar FETs, nanosheet transistors can include a gate stack surrounding the entire periphery of multiple nanosheet channel regions. The nanosheet transistor configuration allows for more complete depletion and reduced short-channel effects within the nanosheet channel regions. A “nanowire transistor” can be similar to a nanosheet transistor, except that the channel can include nanowires instead of nanosheets. The gate-all-around structure in nanosheet or nanowire transistors can provide very small devices with better switching control, lower leakage current, faster operation, and lower output resistance.

[0208] One way to increase channel conductivity and reduce FET size is to form the channel as a nanostructure. For example, a gate-all-around (GAA) nanosheet FET is an architecture used to provide a relatively small FET footprint by forming the channel region as a series of nanosheets. In a GAA configuration, the nanosheet-based FET includes a source region, a drain region, and a stacked nanosheet channel between the source and drain regions. The gate surrounds the stacked nanosheet channel and modulates the electron flow through the nanosheet channel between the source and drain regions. GAA nanosheet FETs 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 embodiments, the channel nanosheets of a p-FET can be SiGe or Si, and the sacrificial nanosheets can be Si or SiGe. Alternating layers of channel nanosheets formed from first-type semiconductor materials (e.g., Si for n-type FETs and SiGe for p-type FETs) and sacrificial nanosheets formed from second-type semiconductor materials (e.g., SiGe for n-type FETs and Si for p-type FETs) provide excellent channel electrostatic control, which is beneficial for continuously scaling gate lengths to seven-nanometer CMOS technology and below. Using multilayer SiGe / Si sacrificial / channel nanosheets (or Si / SiGe sacrificial / channel nanosheets) to form the channel region in a GAA FET semiconductor device provides desired device characteristics, including introducing strain at the interface between SiGe and Si.

[0209] In some examples, "nanowires" are characterized by a critical size of less than about 30 nm, while "nanofashelves" are characterized by a critical size of about 30 nm or larger. In an exemplary device, the critical size is measured along the gate. In this direction, if the channel width is small, the channel cross-section resembles a "wire," while if the channel width is large, the channel cross-section resembles a "sheet."

[0210] In some examples, the minimum size of the nanosheets or nanowires is between approximately 1 nm-10 nm, approximately 1 nm-50 nm, approximately 1 nm-100 nm, approximately 1 nm-500 nm, or approximately 1 nm-1000 nm. In other examples, the minimum size of the nanosheets or nanowires is between approximately 1 nm-5 nm, approximately 3 nm-10 nm, approximately 5 nm-15 nm, approximately 10 nm-20 nm, approximately 15 nm-30 nm, approximately 20 nm-40 nm, approximately 30 nm-50 nm, approximately 40 nm-75 nm, approximately 50 nm-100 nm, approximately 75 nm-150 nm, approximately 100 nm-200 nm, approximately 150 nm-300 nm, approximately 200 nm-400 nm, approximately 300 nm-500 nm, approximately 400 nm-750 nm, or approximately 500 nm-1000 nm. In some examples, the smallest size of the nanosheet is at least about 3 times, about 5 times, about 7 times, about 10 times, about 15 times, about 20 times, about 50 times, about 100 times, about 150 times, about 200 times, about 250 times, about 300 times, about 350 times, about 400 times, about 450 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 2000 times, about 2500 times, about 3000 times, about 4000 times, or about 5000 times smaller than the other two sizes of the nanosheet. In some examples, the smallest size of the nanosheet is approximately 2-5 times, 3-7 times, 5-10 times, 7-15 times, 10-20 times, 15-50 times, 20-100 times, 50-150 times, 100-200 times, 150-250 times, 200-300 times, 250-350 times, and 300-40 times smaller than the other two sizes of the nanosheet. 0x, approximately 350x-450x, approximately 400x-500x, approximately 450x-600x, approximately 500x-700x, approximately 600x-800x, approximately 700x-900x, approximately 800x-1000x, approximately 900x-2000x, approximately 1000x-2500x, approximately 2000x-3000x, approximately 2500x-4000x, or approximately 3000x-5000x. In some examples, the smallest size of the nanosheet is at most about 3 times, about 5 times, about 7 times, about 10 times, about 15 times, about 20 times, about 50 times, about 100 times, about 150 times, about 200 times, about 250 times, about 300 times, about 350 times, about 400 times, about 450 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 2000 times, about 2500 times, about 3000 times, about 4000 times, or about 5000 times smaller than the other two sizes of the nanosheet.In some examples, the largest size of the nanowire is at least about 3 times, about 5 times, about 7 times, about 10 times, about 15 times, about 20 times, about 50 times, about 100 times, about 150 times, about 200 times, about 250 times, about 300 times, about 350 times, about 400 times, about 450 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 2000 times, about 2500 times, about 3000 times, about 4000 times, or about 5000 times larger than the other two sizes of the nanowire. In some examples, the largest size of the nanowire is approximately 2-5 times, 3-7 times, 5-10 times, 7-15 times, 10-20 times, 15-50 times, 20-100 times, 50-150 times, 100-200 times, 150-250 times, 200-300 times, 250-350 times, and 300-40 times larger than the other two sizes of the nanowire. 0x, approximately 350x-450x, approximately 400x-500x, approximately 450x-600x, approximately 500x-700x, approximately 600x-800x, approximately 700x-900x, approximately 800x-1000x, approximately 900x-2000x, approximately 1000x-2500x, approximately 2000x-3000x, approximately 2500x-4000x, or approximately 3000x-5000x. In some examples, the largest size of the nanowire is up to approximately 3, 5, 7, 10, 15, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 2500, 3000, 4000, or 5000 times larger than the other two sizes of the nanowire.

[0211] As used herein, "porous structure" or "glass frit" refers to a bulk material having porous portions. Typical pore sizes can be, for example, about 100 μm or smaller, about 50 μm or smaller, about 10 μm or smaller, about 5 μm or smaller, about 1 μm or smaller, about 500 nm or smaller, about 100 nm or smaller, about 50 nm or smaller, about 10 nm or smaller, about 5 nm or smaller, about 1 nm or smaller, about... or smaller, approximately or smaller, approximately or smaller, approximately or smaller, approximately or smaller, approximately 100 μm or larger, approximately 50 μm or larger, approximately 10 μm or larger, approximately 5 μm or larger, approximately 1 μm or larger, approximately 500 nm or larger, approximately 100 nm or larger, approximately 50 nm or larger, approximately 10 nm or larger, approximately 5 nm or larger, approximately 1 nm or larger, approximately or larger, approximately or larger, approximately or larger, approximately or larger, approximately Or larger, between approximately 500μm and approximately 100μm, between approximately 250μm and approximately 50μm, between approximately 125μm and approximately 25μm, between approximately 50μm and approximately 10μm, between approximately 25μm and approximately 5μm, between approximately 12.5μm and approximately 2.5μm, between approximately 5.5μm and approximately 0.5μm, between approximately 500μm and approximately 100nm, between approximately 250μm and approximately 50nm, between approximately 125μm and approximately 25nm, between approximately 50μm and approximately 10nm, between approximately 25μm and approximately 5nm, between approximately 12.5μm and approximately 2.5nm, between approximately 5.5μm and approximately 0.5nm, between approximately 500μm and approximately Between, approximately 250μm and approximately Between, approximately 125μm and approximately Between, approximately 50μm and approximately Between, approximately 25μm and approximately Between, approximately 12.5 μm and approximately Between, or approximately 5.5 μm and approximately Different pore sizes can exist between them.

[0212] Porous structures can be formed from porous materials comprising a matrix defining an array of pores having a porosity sufficient to achieve the desired functionality of the porous material. As used herein, the term "porosity" refers to the amount of vacant space in a porous material containing a matrix. Therefore, the total volume of a porous material containing a matrix is ​​based on both matrix space and vacant space. As used herein, the term "vacant space" refers to the actual or physical space in a porous material containing a matrix. Therefore, the total volume of a porous material containing a matrix disclosed herein is based on both matrix space and vacant space. For example, a porous material comprising a matrix with a defined pore array can have porosities such as: about 40% of the total matrix volume, about 50% of the total matrix volume, about 60% of the total matrix volume, about 70% of the total matrix volume, about 80% of the total matrix volume, about 90% of the total matrix volume, about 95% of the total matrix volume, or about 97% of the total matrix volume, at least about 40% of the total matrix volume, at least about 50% of the total matrix volume, at least about 60% of the total matrix volume, at least about 70% of the total matrix volume, at least about 80% of the total matrix volume, at least about 90% of the total matrix volume, at least about 95% of the total matrix volume, or at least about 97% of the total matrix volume, at most about 40% of the total matrix volume, at most about 50% of the total matrix volume, at most about 60% of the total matrix volume, at most about 70% of the total matrix volume, at most about 80% of the total matrix volume, or at most about 90% of the total matrix volume. Up to approximately 95% or up to approximately 97% of the total matrix volume; approximately 40% to approximately 97% of the total matrix volume; approximately 50% to approximately 97% of the total matrix volume; approximately 60% to approximately 97% of the total matrix volume; approximately 70% to approximately 97% of the total matrix volume; approximately 80% to approximately 97% of the total matrix volume; approximately 90% to approximately 97% of the total matrix volume; approximately 40% to approximately 95% of the total matrix volume; approximately 50% to approximately 97% of the total matrix volume. Approximately 95%, approximately 60% to approximately 95% of the total matrix volume, approximately 70% to approximately 95% of the total matrix volume, approximately 80% to approximately 95% of the total matrix volume, approximately 90% to approximately 95% of the total matrix volume, approximately 40% to approximately 90% of the total matrix volume, approximately 50% to approximately 90% of the total matrix volume, approximately 60% to approximately 90% of the total matrix volume, approximately 70% to approximately 90% of the total matrix volume, or approximately 80% to approximately 90% of the total matrix volume.For example, a porous material comprising a matrix with a defined pore array may have void spaces of, for example, about 50% of the total matrix volume, about 60% of the total matrix volume, about 70% of the total matrix volume, about 80% of the total matrix volume, about 90% of the total matrix volume, about 95% of the total matrix volume, or about 97% of the total matrix volume, at least about 50% of the total matrix volume, at least about 60% of the total matrix volume, at least about 70% of the total matrix volume, at least about 80% of the total matrix volume, at least about 90% of the total matrix volume, at least about 95% of the total matrix volume, or at least about 97% of the total matrix volume, at most about 50% of the total matrix volume, at most about 60% of the total matrix volume, at most about 70% of the total matrix volume, at most about 80% of the total matrix volume, or the total matrix volume of... Up to about 90%, up to about 95% of the total matrix volume, or up to 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 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.

[0213] Porous structures can be porous matrices, porous membranes, certain types of ion-permeable ionomers, porous glass frits, ion-selective membranes, ion-conducting glasses, polymer membranes, or ion-conducting membranes. Porous structures can be formed from microporous materials (such as ceramic or glass frits, ceramic or glass membranes) or solid porous substrates (such as glass frits or wafers prepared from polymers or inorganic materials). Glass frits can 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.

[0214] In some examples, the porous structure may comprise a microporous membrane formed of polysulfone, polyethersulfone, or polyvinylidene fluoride. In some examples, 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. Furthermore, a hollow fiber membrane in a laminated structure having both a non-porous membrane and a porous membrane can be used, the porous membrane being provided to hold the non-porous membrane therebetween. In some examples, the porous structure can be formed from PEDOT:PSS (poly(3,4-ethylenedioxythiophene)polystyrene sulfonate), silicone, polytetrafluoroethylene, polyethylene-co-tetrafluoroethylene, polyolefins, polyesters, polycarbonates, bio-stabilized polytetrafluoroethylene, homopolymers, copolymers, terpolymers of polyurethanes, polypropylene (PP), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), polymethyl methacrylate (PMMA), polyether ether ketone (PEEK), polyurethanes, cellulose polymers, polysulfones, and their block copolymers (including, for example, diblock, triblock, alternating, random, and graft copolymers).

[0215] In some examples, the porous structure can be formed from porous silica, organosilicon glass (carbon-doped oxide), indium tin oxide (ITO), or a low-κ (low dielectric constant) dielectric, including boron carbide silicon carbon nitride (SiCBN), silicon carbide carbon nitride (SiOCN), fluorine-doped silica, carbon-doped silica, 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. TM BDIIx from AMAT TM And Aurora from ASMi TM Alternative materials can be deposited via spin coating—such materials include SiLK from Dow Chemical. TM And LKD from JSR TMFor example, low-κ porous organosilicon glasses can have a dielectric constant of about 2.7 and a porosity greater than 10% (defined as the volume of the pores divided by the total volume including the material between the pores and the material between the pores). For example, porous silica can have a porosity between about 15% and 40% or between about 30% and 35%. Porous silica can have a porosity that follows... <100> The configuration of vertical and horizontal pores in the crystalline orientation of silicon. Porous silica can be formed from a substrate material, such as porous silicon. In some examples, the porous structure can be formed from a porous material formed by porousification. In some examples, the porous material can be a nanoporous material, that is, pores with a size or diameter in the nanometer range. Porous materials formed by porousification can be provided with pores of small diameters, for example, between about 2 nm and about 100 nm. Porous materials formed by porousification can be fabricated to have an open porosity greater than 30%. In some examples, the porous structure can be formed by porous materials formed through the 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, organosilicon glass (SiCOH), silicon oxide, boron nitride and silicon oxynitride.

[0216] In view of the above definitions, the aspects and examples set forth herein and listed in the claims are to be understood.

[0217] Additional notes

[0218] It should be understood that all combinations of the foregoing concepts and the additional concepts discussed in more detail below (assuming such concepts do not contradict each other) are contemplated as part of the inventive subject matter disclosed herein. Specifically, all combinations of the 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 terms expressly adopted herein that may also appear in any disclosure incorporated by reference should be given the meaning most consistent with the specific concepts disclosed herein.

[0219] Throughout this specification, references to “an example,” “another example,” “a kind of example,” etc., mean that a particular element (e.g., a feature, structure, and / or characteristic) described in connection with that example is included in at least one example described herein and may or may not be present in other examples. Furthermore, it should be understood that the elements used in any example may be combined in any suitable manner across various examples, unless the context clearly indicates otherwise.

[0220] It should be understood that the ranges provided herein include the specified range and any values ​​or subranges within the specified range, as if such values ​​or subranges were explicitly listed. For example, the range of about 2 nm to about 20 nm should be interpreted as including not only the explicitly listed limits of about 2 nm to about 20 nm, but also individual values ​​such as about 3.5 nm, about 8 nm, about 18.2 nm, etc., and subranges such as about 5 nm to about 10 nm, etc. Furthermore, when “about” and / or “substantially” are used to describe values, this means that minute variations (at most + / - 10%) of the stated values ​​are included.

[0221] Although several examples have been described in detail, it should be understood that modifications can be made to the disclosed examples. Therefore, the above description should be considered non-limiting.

[0222] Although certain examples have been described, these examples are presented by way of illustration only and are not intended to limit the scope of this disclosure. In fact, the novel methods and systems described herein can be embodied in many other forms. Furthermore, various omissions, substitutions, and changes can be made to the systems and methods described herein without departing from the spirit of this disclosure. The appended claims and their equivalents are intended to cover such forms or modifications that fall within the scope and spirit of this disclosure.

[0223] Features, materials, properties, or groups described in connection with a particular aspect or example shall be construed as applicable to any other aspect or example described in this section or elsewhere in this specification, unless incompatible with it. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. Protection is not limited to the details of any of the foregoing examples. Protection extends to any novel feature or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel step or any novel combination of steps in any method or process so disclosed.

[0224] Furthermore, certain features described in the context of a single embodiment in this disclosure may also be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment may also be implemented separately in multiple embodiments or in any suitable sub-combination. Moreover, although features may be described as functioning in some combinations, in certain cases, one or more features from the claimed combination may be removed from the combination, and the combination may be claimed as a sub-combination or a variation of a sub-combination.

[0225] Furthermore, while operations may be depicted in the accompanying drawings or described in the specification in a specific order, such operations need not be performed in the specific order shown or sequentially, or all operations need not be performed to obtain the desired result. Other operations not depicted or described may be incorporated into the exemplary methods and processes. For example, one or more additional operations may be performed before, after, simultaneously with, or between any described operations. Furthermore, operations may be rearranged or reordered in other embodiments. Those skilled in the art will understand that in some examples, the actual steps taken in the illustrated and / or disclosed processes may differ from the actual steps shown in the figures. Depending on the example, some of the above steps may be removed, or other steps may be added. Furthermore, the features and properties of the specific examples disclosed above may be combined in different ways to form additional examples, all of which fall within the scope of this disclosure. Moreover, the separation of various system components in the above embodiments should not be construed as requiring such separation in all embodiments; rather, it should be understood that the described components and systems may generally be integrated together in a single product or packaged in multiple products. For example, any component of the energy storage system described herein may be provided separately or integrated together (e.g., packaged together, or attached together) to form an energy storage system.

[0226] For the purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not all of these advantages can necessarily be realized according to any particular example. Thus, for example, those skilled in the art will recognize that this disclosure may be embodied or performed in a manner as taught herein, without necessarily realizing other advantages as taught or suggested herein.

[0227] Unless otherwise specifically stated or otherwise understood within the context in which they are used, conditional language such as "can / could" or "might / may" is generally intended to convey that certain examples include certain features, elements, and / or steps, while other examples do not. Therefore, such conditional language generally does not imply that one or more examples require features, elements, and / or steps in any way, or that one or more examples must include logic for making decisions with or without user input or prompts, regardless of whether the features, elements, and / or steps are included in a particular example or will be performed in any particular example.

[0228] Unless otherwise specified, connective language such as the phrase “at least one of X, Y, and Z” is generally understood in context to convey that an item, term, etc., can be X, Y, or Z. Therefore, such connective language is not usually intended to imply that certain examples require the presence of at least one of X, at least one of Y, and at least one of Z.

[0229] The degree language used in this article, such as the terms “approximately,” “about,” “generally,” and “basically,” indicates a value, quantity, or characteristic that is close to the stated value, quantity, or characteristic and still performs the expected function or achieves the expected result.

[0230] The scope of this disclosure is not intended to be limited by the specific disclosure of the preferred examples in this section or elsewhere in this specification, and may be defined by the claims presented or to be presented elsewhere in this section or elsewhere in this specification. The language of the claims shall be interpreted broadly based on the language used in the claims and is not limited to the examples described in this specification or during the examination of the application, which shall be interpreted as non-exclusive.

Claims

1. An apparatus including a field-effect transistor (FET), the apparatus comprising: intermediate trap; A cis-electrode associated with a cis-electrode, wherein a first nanoscale opening is disposed between the cis-electrode and the intermediate well; An inverted well associated with an inverted electrode, wherein a second nanoscale opening is disposed between the inverted well and the intermediate well; and The FET, located between the first nanoscale opening and the second nanoscale opening, comprises: A source, a drain, and a channel connecting the source to the drain, wherein the source, the drain, and the channel are vertically stacked, and wherein the channel includes a gate oxide layer having a vertical surface fluidly exposed to the intermediate well. The intermediate trap fluidly connects the cis trap to the inverse trap, and The vertical direction is from the cis electrode to the inverse electrode, and the intermediate trap includes a fluid tunnel that does not extend through the channel.

2. An apparatus including a field-effect transistor (FET), the apparatus comprising: intermediate trap; A cis-electrode associated with a cis-electrode, wherein a first nanoscale opening is disposed between the cis-electrode and the intermediate well; An inverted well associated with an inverted electrode, wherein a porous structure is disposed between the inverted well and the intermediate well; and The FET, located between the first nanoscale opening and the porous structure, comprises: A source, a drain, and a channel connecting the source to the drain, wherein the source, the drain, and the channel are vertically stacked, and wherein the channel includes a gate oxide layer having a vertical surface fluidly exposed to the intermediate well. The intermediate trap fluidly connects the cis trap to the inverse trap, and The vertical direction is from the cis electrode to the trans electrode, and the intermediate trap includes a fluid tunnel that does not extend through the channel.

3. The device according to claim 1 or 2, wherein the FET is a nanowire transistor.

4. The device according to claim 1 or 2, wherein the FET is a nanosheet transistor.

5. The apparatus of claim 1 or 2, 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 intermediate well.

6. The apparatus of claim 2, wherein the porous structure comprises a SiCOH thin film.

7. The apparatus of claim 1 or 2, further comprising a membrane located between the cis-well and the intermediate well, wherein the first nanoscale opening extends through the membrane.

8. The apparatus of claim 7, wherein the membrane is selected from the group consisting of lipids and biomimetic equivalents of lipids.

9. The apparatus of claim 7, wherein the first nanoscale opening extends through: a polynucleotide nanopore, a polypeptide nanopore, or a carbon nanotube disposed in the membrane.

10. The apparatus of claim 7, wherein the membrane is a synthetic membrane, and wherein the first nanoscale opening is a solid nanopore.

11. The apparatus of claim 1 or 2, wherein the gate oxide layer has a thickness between 1 nm and 10 nm.

12. The apparatus of claim 1 or 2, wherein the gate oxide layer has a thickness between 2 nm and 4 nm.

13. The apparatus of claim 1 or 2, wherein the apparatus comprises a nanopore sequencer.

14. The apparatus of claim 13, wherein the apparatus comprises an array of a plurality of the nanopore sequencers, wherein: Each of the multiple nanopore sequencers shares a common cis electrode and a common trans electrode. or Each of the multiple nanopore sequencers has a different cis electrode and a different trans electrode; or Each of the multiple nanopore sequencers shares a common cis electrode and has a different trans electrode; or Each of the multiple nanopore sequencers has a different cis electrode and shares a common trans electrode.

15. A method of using an apparatus comprising a field-effect transistor (FET) according to claim 1 or 2, the method comprising: Electrolytes are introduced into each of the cis trap, the inverse trap, and the intermediate trap of the device; A bias voltage is applied between the cis electrode and the trans electrode, wherein the resistance of the first nanoscale opening changes in response to the base identity in the polynucleotide at the first nanoscale opening, and wherein the potential of the electrolyte in the intermediate trap changes in response to the change in the resistance of the first nanoscale opening. as well as The response of the FET is measured as a base function in the polynucleotide at the first nanoscale opening to identify the bases in the polynucleotide.

16. The method of claim 15, wherein measuring the response of the FET comprises measuring: Source-drain current; or The potential at the source, the drain, or both the source and the drain; or The resistance of the channel; or Any combination of them.

17. An apparatus including a field-effect transistor (FET), the apparatus comprising: intermediate trap; A cis-electrode associated with a cis-electrode, wherein a first nanoscale opening is disposed between the cis-electrode and the intermediate well; An inverted well associated with an inverted electrode, wherein a second nanoscale opening is disposed between the inverted well and the intermediate well; and The FET, located between the first nanoscale opening and the second nanoscale opening, comprises: A source, a drain, and a channel connecting the source to the drain, wherein the channel includes a gate oxide layer operatively connected to a metal structure. The gate oxide layer thereon is not fluidly exposed. The intermediate trap fluidly connects the cis trap to the inverse trap, and The metal structure thereon has at least one surface that is fluidly exposed to the intermediate well.

18. An apparatus including a field-effect transistor (FET), the apparatus comprising: intermediate trap; A cis-electrode associated with a cis-electrode, wherein a first nanoscale opening is disposed between the cis-electrode and the intermediate well; An inverted well associated with an inverted electrode, wherein a porous structure is disposed between the inverted well and the intermediate well; and The FET, located between the first nanoscale opening and the porous structure, comprises: A source, a drain, and a channel connecting the source to the drain, wherein the channel includes a gate oxide layer operatively connected to a metal structure. The gate oxide layer thereon is not fluidly exposed. The intermediate trap fluidly connects the cis trap to the inverse trap, and The metal structure thereon has at least one surface that is fluidly exposed to the intermediate well.

19. The apparatus of claim 17 or 18, wherein the metal structure has at least one partial vertical surface, at least two partial vertical surfaces, at least one partial horizontal surface, at least two partial horizontal surfaces, or any combination thereof fluidly exposed to the intermediate trap, wherein the vertical direction is from the cis electrode to the inverse electrode, and wherein the horizontal direction is orthogonal to the vertical direction.

20. The apparatus of claim 17 or 18, wherein the metal structure has at least one cup-shaped structure fluidly exposed to the intermediate trap.

21. The apparatus of claim 17 or 18, wherein the portion of the metal structure fluidly exposed to the intermediate trap includes at least one hole or opening.

22. The apparatus of claim 17 or 18, wherein the portion of the metal structure fluidly exposed to the intermediate trap comprises at least two holes or openings.

23. The device of claim 17 or 18, wherein the FET is a nanowire transistor.

24. The apparatus of claim 23, 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, wherein the length is at least 10 times the width or the height.

25. The device of claim 17 or 18, wherein the FET is a nanosheet transistor.

26. The apparatus of claim 25, wherein the channel has a length along a direction from the source to the drain, a height along a direction 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 5 times the height, and wherein the width is at least 5 times the height.

27. The apparatus of claim 18, wherein the porous structure comprises a SiCOH thin film.

28. The apparatus of claim 17 or 18, further comprising a membrane located between the cis-well and the intermediate well, wherein the first nanoscale opening extends through the membrane.

29. The apparatus of claim 28, wherein the membrane is selected from the group consisting of lipids and biomimetic equivalents of lipids.

30. The apparatus of claim 28, wherein the first nanoscale opening extends through: a polynucleotide nanopore, a polypeptide nanopore, or a carbon nanotube disposed in the membrane.

31. The apparatus of claim 28, wherein the membrane is a synthetic membrane, and wherein the first nanoscale opening is a solid nanopore.

32. The apparatus of claim 17 or 18, wherein the gate oxide layer has a thickness between 1 nm and 10 nm.

33. The apparatus of claim 17 or 18, wherein the gate oxide layer has a thickness between 2 nm and 4 nm.

34. The apparatus of claim 17 or 18, wherein the apparatus comprises a nanopore sequencer.

35. The apparatus of claim 17 or 18, wherein the at least one surface of the fluidly exposed metal structure is formed of a corrosion-resistant material.

36. The apparatus of claim 34, wherein the apparatus comprises an array of a plurality of the nanopore sequencers, wherein: Each of the multiple nanopore sequencers shares a common cis electrode and a common trans electrode. or Each of the multiple nanopore sequencers has a different cis electrode and a different trans electrode; or Each of the multiple nanopore sequencers shares a common cis electrode and has a different trans electrode; or Each of the multiple nanopore sequencers has a different cis electrode and shares a common trans electrode.

37. A method of using an apparatus comprising a field-effect transistor (FET) according to claim 17 or 18, the method comprising: Electrolytes are introduced into each of the cis trap, the inverse trap, and the intermediate trap of the device; A bias voltage is applied between the cis electrode and the trans electrode, wherein the resistance of the first nanoscale opening changes in response to the base identity in the polynucleotide at the first nanoscale opening, and wherein the potential of the electrolyte in the intermediate trap changes in response to the change in the resistance of the first nanoscale opening. as well as The response of the FET is measured as a base function in the polynucleotide at the first nanoscale opening to identify the bases in the polynucleotide.

38. The method of claim 37, wherein measuring the response of the FET comprises measuring: Source-drain current; or The potential at the source, the drain, or both the source and the drain; or The resistance of the channel; or Any combination of them.

39. The method of claim 37, wherein no electrochemical reaction occurs at the at least one fluidly exposed surface of the metal structure.

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