3D nanopore device

Abstract

To provide a system and a device for determining a nanopore-based arrangement having an acceptable sensitivity and manufacturing cost.SOLUTION: A 3D nanopore device for characterizing a biopolymer molecule includes a first selection layer having a first selection axis. The device is arranged next to the first selection layer and includes a second selection layer having a second selection axis perpendicular to the first selection axis. The device further includes a third electrode layer arranged next to the second selection layer. The first, second, and third electrode layers form a stack of layers along a Z-axis and define a plurality of nanopore pillars.SELECTED DRAWING: Figure 2-1

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE

[0001] This disclosure relates generally to systems, devices, and processes for characterizing biopolymer molecules, as well as methods for manufacturing such systems and devices. [Background technology]

[0002] Nucleic acid (e.g., DNA, RNA) sequencing is one of the most powerful methods for identifying genetic variations at the molecular level. Many genetic disease characteristics can be diagnosed through information gathered through genome-wide single nucleotide polymorphism ("SNP") analysis, gene fusions, genomic insertions and deletions, and other molecular biology techniques. These and other molecular biology techniques require nucleic acid sequencing at some point. Current technologies for sequencing nucleic acids at the single-molecule level include nanopore sequencing, which offers label-free and amplification-free technology characteristics, improved read lengths, and improved system throughput. Therefore, nanopore sequencing is being incorporated into high-quality gene sequencing applications.

[0003]

[0003] Early experimental systems for nanopore-based DNA sequencing detected the electrical behavior of ssDNA passing through an α-hemolysin (αHL) protein nanopore. Since then, nanopore-based nucleic acid sequencing techniques have improved. For example, as described below, solid-state nanopore-based nucleic acid sequencing replaces biological / protein-based nanopores with solid-state (e.g., semiconductor, metal-gate) nanopores.

[0004]

[0004] Nanopores are small holes (e.g., holes with diameters of about 1 nm to about 1000 nm) that can detect the flow of electrons through the hole by changes in ionic and / or tunneling current. Each nucleotide of a nucleic acid (e.g., adenine, cytosine, guanine, thymine in DNA, and uracil in RNA) affects the current density across the nanopore in a specific way as it physically passes through the nanopore, and measuring the changes in current through the nanopore during translocation provides data that can be used to directly sequence nucleic acid molecules passing through the nanopore. Thus, nanopore technology is based on electrical sensing and can detect nucleic acid molecules at concentrations and volumes much smaller than those required for other conventional sequencing methods. Advantages of nanopore-based nucleic acid sequencing include long read lengths, plug-and-play capabilities, and scalability. However, current biological nanopore-based nucleic acid sequencing technologies require fixed nanopore openings (e.g., with diameters of approximately 2 nm), suffer from insufficient sensitivity (i.e., unacceptable amounts of false negatives), are prohibitively expensive to manufacture, and are highly dependent on temperature and concentration (e.g., pH).

[0005]

[0005] With advances in semiconductor fabrication technology, solid-state nanopores have become a cheaper and superior alternative to biological nanopores due to their superior mechanical, chemical, and thermal properties, as well as their compatibility with semiconductor technology, which allows for integration with other sensing circuits and nanodevices. However, current nanopore DNA sequencing technologies (including biological and / or solid-state nanopores) continue to suffer from various limitations, including low sensitivity and high manufacturing costs. Figure 1 shows a schematic diagram of a state-of-the-art solid-state-based two-dimensional ("2D") nanopore sequencing device 100. Although the device 100 is called "2D," the device 100 has some thickness along the Z-axis.

[0006]

[0006] Many of the limitations of nanopore DNA sequencing technology stem from the intrinsic nature of nanopore devices and the techniques required to overcome the high translocation speed and small size (e.g., height of approximately 0.34 nm and diameter of approximately 1 nm) of single nucleotides. Conventional electronics (e.g., nanoelectrodes) cannot analyze such fast-translocating small nucleotides using conventional nanopore-based DNA sequencing technologies. Additionally, high manufacturing costs hinder the widespread application of nanopore-based DNA sequencing.

[0007] Many efforts have been made to overcome these shortcomings, including the use of many different types of biological, solid-state, and hybrid (biological and solid-state) nanopores and nanopore sensors, but none of these efforts have been successful in mass production.

[0008] There is a need for nanopore-based sequencing systems and devices that address the shortcomings of currently available configurations, and in particular, there is a need for nanopore-based sequencing systems and devices that have acceptable sensitivity and manufacturing costs. Summary of the Invention

[0009]

[0009] Embodiments described herein are directed to nanopore-based sequencing systems and methods of making same. In particular, embodiments relate to 3D nanopore-based sequencing systems and methods of making same.

[0010] In one embodiment, a 3D nanopore device for characterizing biopolymer molecules includes a first selective layer having a first selective axis. The device also includes a second selective layer disposed adjacent to the first selective layer and having a second selective axis orthogonal to the first selective axis. The device further includes a third electrode layer disposed adjacent to the second selective layer, wherein the first selective layer, the second selective layer, and the third electrode layer form a stack of layers along the Z axis and define a plurality of nanopore pillars.

[0011] In one or more embodiments, the first selective layer includes a first plurality of suppression electrodes. The second selective layer may include a second plurality of suppression electrodes. The first and second plurality of suppression electrodes may form an array that partially defines a plurality of nanopore pillars therein. The third electrode layer may include electrodes configured to modulate the electrical bias and detect current modulation. The device may also include one or more electrode layers disposed adjacent to the third electrode layer.

[0012] In one or more embodiments, the device also includes an upper chamber disposed adjacent to the first selective layer. The device further includes a lower chamber disposed adjacent to the lower electrode layer, and when multiple electrodes are present in the device, the plurality of nanopore pillars fluidly couple the upper and lower chambers and provide a translocation channel. The device can also include an electrolyte within the upper and lower chambers and surrounding the first selective layer, the second selective layer, and the third electrode layer. The electrolyte can include KCl or LiCl2.

[0013] In one or more embodiments, the third electrode layer includes a metal velocity control electrode. The third electrode layer may include a metal such as Ta, Al, Cr, Au-Cr, Ti, graphene, or Al-Cu. The third electrode may include highly doped (n+ or p+ type) polysilicon or salicide polysilicon. The third electrode layer may have a thickness of 0.2 nm to 1000 nm. The third electrode layer may include a sensing electrode. The sensing electrode may operate by ion blockage, tunneling, capacitive sensing, piezoelectric, or microwave sensing.

[0014] In one or more embodiments, the device also includes an inner membrane layer configured to modify the inner diameter of the plurality of nanopores. The inner membrane layer can include a low-stress silicon-rich nitride, such as Si3N4, and is coated with a dielectric, such as Al2O3, SiO2, ZnO, or HfO2. The inner membrane layer can have a thickness of about 10 nm to about 50 nm. Each of the plurality of nanopores can have a respective diameter of about 0.2 nm to about 1000 nm. The device can also include an upper membrane layer. The upper membrane layer can include Si3N4, Al2O3, SiO2, a 2D dielectric (e.g., MoS2 or hBN), and a polymer membrane (e.g., polyimide and PDMS). The upper membrane layer can have a thickness of about 5 nm to about 50 nm.

[0015] In another embodiment, a method for fabricating a 3D nanopore device includes depositing a first Si3N4 layer on a first Si substrate or first dielectric base layer. The method includes depositing a first dielectric layer on the first Si3N4 layer. The method also includes depositing a first metal or polysilicon layer on the first dielectric layer. In one or more embodiments, the method also includes etching and patterning the first metal or polysilicon electrode layer. The method also includes depositing a second dielectric layer on the patterned first metal or polysilicon electrode layer.

[0016] The method also includes depositing a second metal or polysilicon layer on the first metal or polysilicon electrode layer. The method further includes depositing a second Si3N4 layer on the second dielectric layer. In one or more embodiments, the method also includes etching and patterning the second metal or polysilicon electrode layer. The method further includes depositing and patterning multiple layers of metal or polysilicon electrode layers.

[0017] The method includes etching a first Si or dielectric substrate base layer from the backside to create a channel from the backside.

[0018] The method includes patterning nanopore channels from a surface on a plurality of stacks of Si3N4 layers, dielectric layers, and metal or polysilicon layers to form nanopores therethrough. The method can also include disposing a respective metal or polysilicon electrode in every channel and electrically coupling the metal or polysilicon electrode layers.

[0019] In one or more embodiments, the method also includes etching a second channel into the bottom Si3N4 layer, where the first and second channels are orthogonal to one another. The method may also include disposing a second suppression electrode in the second channel and electrically coupling the second suppression electrode to the bottom Si3N4 layer. The method may also include depositing a third dielectric base layer on the second metal layer and etching the third dielectric base layer to form a nanopore therethrough. The method may also include etching the third dielectric layer and electrically coupling the third electrode to the third dielectric layer. The method may also include etching the substrate and fluidically coupling the lower chamber to the plurality of nanopore pillars.

[0020] In one or more embodiments, the method also includes depositing a first dielectric base layer, a first Si3N4 layer, and a first metal in an intermediate chamber between the upper and lower chambers, where the upper, intermediate, and lower chambers contain an electrolyte, and the upper and lower chambers are fluidly coupled by the nanopore. Deposition of the first Si3N4 layer, the first metal layer, and the dielectric coating layer can utilize ALD or CVD. Etching the first dielectric base layer, the first Si3N4 layer, and the first metal layer to form the nanopore can utilize high-aspect-ratio etching.

[0021] In yet another embodiment, a method for detecting charged particles uses a 3D nanopore device having an upper chamber, an intermediate chamber, and a lower chamber, and a 3D nanopore array disposed in the intermediate chamber such that the upper and lower chambers are fluidly coupled by a plurality of nanopores in the 3D nanopore array. The method includes adding an electrolyte solution containing the charged particles to the upper, intermediate, and lower chambers. The method also includes disposing upper and lower electrodes in the upper and lower chambers, respectively. The method further includes applying an electrophoretic bias between the upper and lower electrodes. The method further includes applying first and second selection biases to the first and second selection electrodes of the 3D nanopore device to select one or more nanopores among the plurality of nanopores to which the charged particles are directed. The method further includes applying a rate-control bias to a rate-control electrode of the 3D nanopore device to adjust the rate of movement of the charged particles through the one or more nanopores. The method also includes applying a sensing bias to a sensing electrode in the 3D nanopore device. The method further includes detecting a change in current in the sensing electrode.

[0022] In one or more embodiments, the current is an electrode current or a tunneling current.

[0023] In yet another embodiment, a method for fabricating a sensor comprising a 3D nanopore channel pillar array, a plurality of electrodes, an upper chamber, and a lower chamber includes disposing the 3D nanopore channel pillar array in an electrolyte solution containing biomolecules and DNA. The method also includes disposing electrodes in the electrolyte. The method further includes applying a bias to the electrodes in the electrolyte. The method further includes disposing suppression electrodes in a crossing pattern over the 3D nanopore channel pillar array, surrounding the nanopore pillars. The method further includes disposing metallic planar electrodes surrounding the nanopore pillars in the 3D nanopore channel pillar array, the metallic planar electrodes including rate-controlling electrodes and sensing electrodes. The method also includes applying a rate-controlling bias to the rate-controlling electrodes. The method further includes applying a sensing bias to the sensing electrode. The method further includes detecting a change in electrode current in the electrolyte. The method further includes detecting a change in tunneling current in the electrodes.

[0024] In one or more embodiments, the rate-controlling electrode has a thickness ranging from about 2 nm to about 1000 nm. The rate-controlling electrode may include Ta, Cr, Al, Au-Cr, graphene, or Al-Cu. It may also include highly doped (n- or p-type) polysilicon or salicide polysilicon. The 3D nanopore channel pillar array may include a biological layer with the rate-controlling electrode to form a hybrid 3D nanopore channel pillar array. The upper and lower chambers may contain at least a portion of an electrolyte. The electrolyte may include KCl and LiCl2. The electrodes in the upper and lower chambers may include Ag / AgCl2. Cross-patterned column and row suppression electrodes may be used to select and deselect columns and rows by applying an suppression bias, thereby enabling array operation to stop vertical ionic current flow. The sensing electrodes may utilize ion-blocking sensing, tunneling sensing, capacitive sensing, piezoelectric sensing, and / or wave sensing.

[0025] In one or more embodiments, the 3D nanopore channel pillar array comprises a plurality of dielectric electrodes in a dielectric electrode stack. The dielectric electrode stack includes a membrane layer, a dielectric layer for varying the nanopore channel opening width, an array of nanopore channel pillars, a stack of rate-controlling dielectric electrode layers, a stack of sensing dielectric electrode layers, and a source-selection dielectric electrode layer. The membrane layer comprises a dielectric material and can have a thickness of about 10 nm to about 50 nm. The dielectric material can be Si3N4, Al2O3, or SiO2. The membrane layer can vary the nanopore channel opening width. The nanopore channel opening width can be about 2 nm to about 100 nm, patterned by standard optical lithography and ion beam (e.g., FIB, TEM) techniques.

[0026]

[0026] In one or more embodiments, the 3D nanopore channel pillar array is fabricated using ALD and / or CVD deposition of dielectric layers, high aspect ratio reactive ion etching deep trench processing (nanopore channel opening etching), ALD and / or CVD deposition of trimming dielectric layers, and / or ALD and / or CVD deposition of membrane dielectric layers.

[0027]

[0027] In one or more embodiments, the dielectric electrode stack also includes a bottom dielectric layer. This bottom dielectric layer may have a thickness of about 100 nm to 1000 nm. The bottom dielectric layer may include SiO2, glass, or SOI to reduce substrate-coupled low-level noise. The dielectric electrode stack may also include a top dielectric layer. The top dielectric layer may have a thickness of about 5 nm to about 50 nm. The top dielectric layer may include SiO2, Si3N4, or Al2O3. The top dielectric layer may determine the final nanopore channel opening width.

[0028] In one or more embodiments, the method also includes forming the nanopore channel pillars using a high aspect ratio etch to impart a sharp shape to the trench profile of the nanopore channel pillars. The high aspect ratio etch can have an aspect ratio greater than 5.

[0029] In one or more embodiments, the 3D nanopore channel pillar array facilitates multiplexed sequencing applications using high-density, low-cost nanopore channels. By selecting the number of electrodes in the 3D nanopore array depending on the desired sequencing application, it is possible to provide time-of-flight ("TOF") technology, where the translocation rate is controlled by a controlled bias. Controlling the translocation rate improves the readout of DNA molecules and increases the sensitivity of the sensor.

[0030] In one or more embodiments, the 3D nanopore channel pillar array is integrated into a CMOS flow, thereby facilitating an implantable biosensor solution for CMOS technology. The CMOS flow may include two-dimensional wells for electrochemical reactions. The CMOS flow may include ion-sensitive field-effect transistor technology.

[0031] In one or more embodiments, the 3D nanopore channel pillar array incorporates hybrid nanopore technology, including biological and solid-state elements in a 3D configuration. The 3D nanopore channel pillar array facilitates electrochemical, thermal, or electro-optical reactions with enlarged individual nanopore wells with a multi-electrode system to enhance electrochemical and sequencing reactions. The 3D nanopore channel pillar array can facilitate multiplexed sequencing using a multi-array configuration in which individual nanopore channel pillars are addressable. The 3D nanopore channel pillar array facilitates standard qPCR within the nanopore channel pillars. The 3D nanopore channel pillar array facilitates probe-mediated targeted sequencing. The 3D nanopore channel pillar array facilitates tuning of nanopore channel opening widths for different applications.

[0032] In one or more embodiments, the nanopore channel opening width is tunable from about 1 nm to about 100 nm. The nanopore channel opening width may be electronically tunable during fabrication.

[0033] In one or more embodiments, the method also includes forming a hybrid nanopore channel to enhance the stability of the sensor. Forming the hybrid nanopore can include inserting a stable biological element to construct a semi-synthetic membrane porin. The stable biological element can be an αHL molecule. The αHL molecule can be inserted into the SiN-based 3D nanopore.

[0034] In one or more embodiments, the method also includes using the upper suppression electrode to introduce structure into the stable biological element to ensure alignment of the stable biological element with the hybrid nanopore.

[0035]

[0035] These and other embodiments of the present disclosure are described in the detailed description below. [Brief explanation of the drawings]

[0036]

[0036] The above and other embodiments will be described in further detail with reference to the accompanying drawings, in which like components in different figures are referred to by common reference numerals. [Figure 1]

[0037] FIG. 1 shows a schematic representation of a prior art solid-state 2D nanopore device. [Figure 2]

[0038] 2A to 2D are a perspective view, a top view, a front view, and a right side view, respectively, that schematically show a 3D nanopore device according to one embodiment. [Figure 3]

[0039] FIG. 3 shows a schematic representation of a 3D nanopore device according to one embodiment, including some details of its operation. [Figure 4]

[0040] FIG. 4 is a table summarizing the voltage behavior of the nanopore device shown in FIG. [Figure 5]

[0041] FIG. 5 is a schematic diagram of a 3D nanopore device according to one embodiment with several electrodes therein. [Figure 6]

[0042] 6A-6E illustrate a method of fabricating a 3D nanopore device according to one embodiment. [Figure 7]

[0043] 7A-7E illustrate a method of fabricating a 3D nanopore device according to another embodiment.

[0044] To better understand how the above and other advantages and objects of the various embodiments are obtained, a more detailed description of the embodiments will be provided with reference to the accompanying drawings. It should be noted that the drawings are not drawn to scale, and that components of similar structure or function are represented by similar reference numerals throughout. It should be understood that these drawings depict only certain illustrated embodiments, and therefore should not be considered as limiting the scope of the embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0037] Exemplary Nanopore Devices

[0045] As discussed above, current state-of-the-art nanopore devices are limited in at least terms of sensitivity and manufacturing cost. The nanopore device embodiments described herein address, among other things, these limitations of current nanopore devices.

[0038]

[0046] 2A-2D are various schematic illustrations of a nanopore device 200 incorporating solid-state nanopore technology with a three-dimensional ("3D") array architecture, according to one embodiment. As shown in FIG. 2A, device 200 comprises multiple 2D arrays or layers 202A-202E stacked along a Z-axis 204. Although the 2D arrays 202A-202E are referred to as "two-dimensional," each 2D array 202A-202E has some thickness along the Z-axis. FIG. 2B shows a top view of the top 2D array 202A shown in FIG. 2A. FIGS. 2C and 2D are schematic illustrations of a front view and a right side view of nanopore device 200 shown in FIG. 2A.

[0039]

[0047] The top 2D array 202A includes first and second selection (suppression electrode) layers 206, 208 configured to direct the movement of charged particles (e.g., biopolymers) through nanopores 210 (pillars) formed in the first and second selection layers 206, 208. The first selection layer 206 is configured to select from multiple rows (R1-R3) within the 2D array 202A. The second selection layer 208 is configured to select from multiple columns (C1-C3) within the 2D array 202A. In one embodiment, the first and second selection layers 206, 208 select from the rows and columns by altering the charge adjacent to the selected rows and columns and / or adjacent to the unselected rows and columns, respectively. The other 2D arrays 202B-202E include velocity control / current sensing electrodes. The velocity control electrodes can be made of a highly conductive metal, such as Au-Cr, TiN, TaN, Pt, Cr, graphene, or Al-Cu. The rate-controlling electrode can have a thickness of about 2 to about 1000 nm. The rate-controlling electrode may also be fabricated in the biological layer within the hybrid nanopore.

[0040]

[0048] The hybrid nanopore comprises a stable biological / biochemical element with a solid element forming a semisynthetic membrane porin to enhance nanopore stability. For example, the biological element can be an αHL molecule. The αHL molecule can be inserted into the SiN-based 3D nanopore. By applying a bias to the electrodes (e.g., in the top 2D array 202A), the αHL molecule can be induced into a structure that ensures alignment of the αHL molecule with the SiN-based 3D nanopore.

[0041]

[0049] Nanopore device 200 has a 3D vertical pillar stack array structure that provides a much larger surface area for charge detection than conventional nanopore devices with planar structures. As charged particles (e.g., biopolymers) pass through each of the 2D arrays 202A-202E within the device, their charges can be detected by detectors (e.g., electrodes) within some of the 2D arrays 202B-202E. Therefore, the 3D array structure of device 200 achieves higher sensitivity, which can compensate for low-signal detectors / electrodes. Furthermore, the highly integrated, small-form-factor 3D structure minimizes manufacturing costs while providing a high-density nanopore array.

[0042]

[0050] In operation, nanopore device 200 is placed in a middle chamber (not shown) that separates the upper and lower chambers such that the upper and lower chambers are fluidly coupled by nanopore pillars 210. The upper, middle, and lower chambers contain an electrolyte solution (e.g., Ag, AgCl) that contains charged particles (e.g., DNA) to be detected. Different electrolyte solutions can be used for the detection of different charged particles.

[0043]

[0051] Translocation of charged particles by electrophoresis can be effected by applying a bias to electrodes disposed in an upper chamber (not shown) adjacent to the top 2D array 202A of the nanopore device 200 and a lower chamber (not shown) adjacent to the bottom 2D array 202E of the nanopore device 200. In some embodiments, the nanopore device 200 is disposed in a middle chamber (not shown) such that the upper and lower chambers are fluidly and electrically coupled by nanopore pillars 210 within the nanopore device 200. The upper, middle, and lower chambers can contain an electrolyte.

[0044]

[0052] FIG. 3 is a schematic diagram of a nanopore device 300 according to another embodiment. It depicts a top 2D array 302 in a cross-sectional (xz plane) view showing 3D nanopores 310 and a nanoelectrode scheme. Each nanopore 310 is surrounded by a nanoelectrode 312, allowing the nanopore 310 channel to operate under a bias electric field generated using the nanoelectrodes 312. Cross-patterned nanogap nanoelectrodes 312CS-312Cn, 312RS-312Rn are arranged in two layers on top of the nanopore device 300. These nanoelectrodes 312CS-312Cn, 312RS-312Rn are the column and row inhibitory nanoelectrodes 312CS-312Cn, 312RS-312Rn for the nanopore array, respectively. The cross-patterned nanoelectrodes 312CS-312Cn, 312RS-312Rn shown in the top 2D array 302 (x-y plan view) can be formed / patterned with a metal lithography step. The remaining 2D arrays of nanoelectrodes 312 in the 3D stack can be formed by planar deposition of metal. The hole pillars of the nanopore 310 are surrounded by the metal nanoelectrodes 312CS-312Cn, 312RS-312Rn and can therefore operate under the full influence of an electrical bias applied to the multiple stacked nanoelectrodes 312.

[0045]

[0053] According to one embodiment, applying an inhibitory electrical bias (0V-VCC) to select nanogap nanoelectrodes 312CS-312Cn, 312RS-312Rn in the top 2D array 302 inhibits biomolecular migration (e.g., electrophoresis) through one or more nanopores 302 in the top 2D nanopore array 302, thereby controlling the operation of the nanopore array. The electrical bias applied to the nanoelectrodes 312CS-312Cn, 312RS-312Rn can generate an electric field sufficient to inhibit ionic migration of charged particles (e.g., nucleic acids) from the upper chamber (not shown) to the lower chamber (not shown) in a direction perpendicular to the nanoelectrodes 312CS-312Cn, 312RS-312Rn. The inhibition of ionic migration through the nanoelectrodes 312 can be substantially complete, or the electrical bias can be adjusted to merely slow the ionic migration rate. In one embodiment, after selecting one or more nanopores 310 (e.g., for translocation and sequencing of DNA biomolecules), the electrical bias on the stack of 3D nanopore nanoelectrodes 312 can be varied to control the translocation rate of the biomolecules. In one embodiment, the inhibitory electrical bias reduces / stops the flow of vertical ionic current, thereby selecting and / or deselecting various columns and rows defined by the nanogap nanoelectrodes 312C-312Cn, 312RS-312Rn. Simultaneously, the nanoelectrodes 312 can detect current changes caused by the passage of charged particles (e.g., DNA biomolecules) through the 3D vertical nanopore 310 pillars. In some embodiments, the nanoelectrodes 312 can detect current changes using various principles, including ion blocking, tunneling, capacitive sensing, piezoelectric, and microwave sensing.

[0046]

[0054] Figure 4 is a table 400 illustrating the voltage operation of the nanopore device 300 shown in Figure 3. As shown in Figure 4, the nanopore device 300 can be operated in both a translocation mode and a read (sensing) mode by modulating the voltages applied to the various electrodes 312.

[0047]

[0055] 5 schematically illustrates a single 3D nanopore sensor 520 within a nanopore device, according to one embodiment. The sensor 520 has a column suppression electrode layer 522, a row suppression electrode layer 524, and multiple rate control / sensing electrode layers 526, 528, 530. These layers are stacked on top of each other and separated by insulator layers 532 (e.g., SiO2) to define a vertical nanopore 510 hole pillar. Each layer has a top sublayer of polysilicon or metal (e.g., Ta, Al, Cr, Au—Cr, Ni, graphene, etc.) and various other sublayers (e.g., Al2O3, Si3N4, n + , or p + The 3D nanopore sensor 520 may have a material selected from the group consisting of silicon, polysilicon, and the like. The 3D nanopore sensor 520 can operate on a variety of principles, including ion blocking, tunneling, capacitive sensing, piezoelectric, and microwave sensing. The rate control / sensing electrode layers 526, 528, 530 can be actuated by applying a rate control or sensing bias to the respective electrode layers 526, 528, 530. The sensing electrode layers 526, 528, 530 can detect changes in electrical properties (e.g., electrode current and / or tunneling current).

[0048]

[0056] Because 2D arrays 202, 302 of 3D nanopore devices 200, 300 can be stacked vertically instead of horizontally, 3D nanopore devices (e.g., 200, 300) enable direct or targeted sequencing within the array while minimizing form factor overhead, thereby enabling high-density applications. Furthermore, 3D nanopore devices (e.g., 200, 300) are scalable to medium- to large-scale 3D nanopore devices with over 1,000 nanopores 210, 310 pillars. As a result, many more sequencing sensors can be accommodated in the same form factor. 3D nanopore devices (e.g., 200, 300) can also incorporate biological or hybrid nanopore technologies, providing a more flexible architecture to accommodate user needs.

[0049]

[0057] In a 3D nanopore device (e.g., 300), each nanopore 310 pillar is composed of a stack of nanoelectrodes 312 that define multiple nanopores 310. Thus, the effective surface area of ​​the sensors within each nanopore 310 array can be several orders of magnitude larger than the surface area of ​​a single sensor. In one embodiment, the sensor effective surface area can be two to three orders of magnitude larger than the surface area of ​​a single sensor. This increased sensor effective surface area can significantly improve the signal-to-noise ratio and sensitivity of the sensor while minimizing manufacturing costs.

[0050] Exemplary Nanopore Device Fabrication Methods

[0058] 3D nanopore devices (e.g., 200, 300) can be fabricated in a variety of ways. In one embodiment, semiconductor technology (e.g., the CMOS process described below) is used to fabricate the 3D nanopore devices 200, 300. The CMOS process also allows for tuning the width of the nanopore 310 using large nanopore arrays. In one embodiment, the width of the nanopore 310 can be controlled during fabrication using software with lookup tables, allowing for mass production manufacturing. The CMOS process allows for biosensor solutions to be integrated into CMOS technology. In various embodiments, the CMOS process includes two-dimensional wells for electrochemical reactions and / or ion-sensitive field-effect transistor technology. Microfluidic channels can be integrated into the 3D nanopore devices 200, 300 (e.g., within the die), thereby reducing the cost of the devices 200, 300.

[0051]

[0059] 6A-6E illustrate a method 600 for fabricating a nanopore device, according to one embodiment. As shown in FIG. 6A, a first dielectric base layer 602A (e.g., SiO2, Al2O3, etc.), a first base layer 604A of Si3N4, and a first metal (e.g., Au-Cr, Al, graphene, etc.) or polysilicon layer 606A are deposited on top of each other. Next, second and third dielectric base, base, and metal layers 602B, 604B, 606B, 602C, 604C, 606C are deposited on top of each other and the previous layers. For example, these deposition steps can be performed using chemical vapor deposition ("CVD") and / or atomic layer deposition ("ALD") of the base dielectric layer 602, trim dielectric layer, and / or membrane dielectric layer (see FIG. 6C below). The first dielectric base layer 602A can have a thickness of approximately 100 nm to 1000 nm to reduce substrate-coupled low-level noise.

[0052]

[0060] As shown in Figure 6B, nanopores 610 are then etched into the deposited layers (e.g., using a high aspect ratio (greater than 5) nanopore hole trench etching process). High aspect ratio etching can provide a sharp shape to the trench profile of the channel pillar of nanopore 610. The total nanopore pillar depth can be from hundreds of nanometers to several microns depending on the application.

[0053]

[0061] Next, as shown in FIG. 6C, a thin layer of dielectric coating 612 (e.g., Si3N4, Al2O3, SiO2, etc.) is deposited (e.g., by atomic layer deposition (ALD)) on the inner surface of nanopore 610 to determine the width of nanopore 610. Dielectric coating 612 can vary in thickness (e.g., from about 10 nm to about 50 nm). By controlling the amount of dielectric coating 612 deposited on the inner surface of nanopore 610 (e.g., using ALD), a target nanopore 610 width of about 2 nm to about 100 nm can be achieved. Thus, the width / diameter of nanopore / trench 610 can be controlled using ALD of dielectric coating 612 to suit various applications. The top dielectric coating 612 can have a thickness of about 5 nm to about 20 nm. Depending on the application and the desired nanopore 610 opening dimensions, various lithography techniques (e.g., those used in mass production) can be used to etch the opening of nanopore 610. Furthermore, the nanopore 610 channel depth can be easily selected with the required sensitivity and precision using the fabrication methods described herein.

[0054]

[0062] As shown in Figure 6D, the vertical nanopore channel and stack layers are etched (see the "steps" to the right of the stack layers) to form horizontal (X-axis) and vertical (Y-axis) nanopore channels providing access to electrodes 614 on the top and addressing circuitry (e.g., row and column suppression electrodes). The base Si3N4 (or Al2O3) layer 604A is selectively wet-etched to allow electrical access to all horizontal electrodes. Finally, the remaining space is filled with metal.

[0055]

[0063] FIG. 6E shows the fabricated 3D nanopore device used for sequencing biopolymers (such as DNA).

[0056]

[0064] Figures 7A-7E show another embodiment of a method 700 for fabricating a nanopore device. The methods 600, 700 shown in Figures 6A-6E and 7A-7E are similar and share many of the same techniques.

[0057]

[0065] As shown in Figure 7A, multilayer dielectric films (SiO2, Al2O3, ZnO, HfO2, etc.; 10 nm to 100 nm), low-stress nitride films (Si3N4), metals (Ta, Al, Cr, Ti, Au-Cr, graphene, etc.; several nm to several hundred nm), and intermetallic layers (SiO2), collectively designated 702, are layer-by-layer deposited on a Si or quartz substrate using CVD (low pressure / plasma enhanced) or atomic layer deposition (ALD). Next, a lower chamber opening 704 (5 × 5 to 100 × 100 μm) is opened. 2 ) is etched by deep reactive ion etching (RIE) or KOH wet etching.

[0058]

[0066] As shown in Figure 7B, a top metal layer 706 is deposited. Next, the upper chamber opening 708 is defined using a high-aspect-ratio nanopore hole deep trench etching process by reactive ion etching. This process can produce nanopore trench etch opening diameters from a few nanometers to approximately 100 nm. Lithography techniques using colloidal masks (e.g., nanodots, quantum dots, graphene oxide pores) can also be used instead of conventional tools.

[0059]

[0067] 7C and 7D, a thin layer (e.g., film) of dielectric 710 may be deposited by ALD to trim the nanopore width to achieve a target nanopore width (using ALD) of about 2 nm to about 1000 nm. The width of the nanopore channel can also be controlled to have a variable trench width, allowing the nanopore width to be varied to match a target diameter.

[0060]

[0068] FIG. 7E shows the fabricated 3D nanopore device used for sequencing biopolymers (such as DNA).

[0061]

[0069] This 3D nanopore device can realize multiplexed sequencing applications using high-density, low-cost nanopore channels. Depending on the desired sequencing application, the number of electrodes in the 3D nanopore device can be selected to provide time-of-flight ("TOF") technology, which controls the translocation speed through controlled bias. Controlling the translocation speed improves the readout of DNA molecules and increases the sensitivity of the sensor. This 3D nanopore device can also facilitate electrochemical, thermal, or electro-optical reactions in enlarged, isolated nanopore wells with a multi-electrode system, enhancing electrochemical and / or sequencing reactions. This 3D nanopore device can further facilitate multiplexed sequencing using a multi-array configuration in which individual nanopore channel pillars are addressable. Furthermore, this 3D nanopore device further facilitates standard qPCR and probe-mediated targeted sequencing within the nanopore channel pillars. Furthermore, the aperture width of the 3D nanopore channel can be tailored to suit various applications. In one embodiment, the nanopore channel aperture width is adjustable from approximately 1 nm to approximately 100 nm. The nanopore channel opening width can be electronically tunable during fabrication. The 3D nanopore devices described herein can be used for the detection (direct detection) of various charged particles, including, but not limited to, biomolecules such as nucleotides, nucleic acids, and proteins. The 3D nanopore devices can also be used for DNA sequencing and protein-DNA interaction detection.

[0062]

[0070] Fabricating metal or polysilicon plane-based nanopore arrays using lithographic processes (e.g., through-silicon via ("TSV") fabrication methods) minimizes fabrication costs and line resistance (thus significantly reducing the IR drop and RC delay limitations on scaling).

[0063]

[0071] The corresponding structure, material, acts, and equivalents of all means or step and functional elements in the following claims are intended to include all such structure, material, acts, and equivalents for performing the function in combination with other claim elements as specifically claimed. While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing description and claims do not limit the scope of the present disclosure. Other advantages and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

[0064]

[0072] Various exemplary embodiments of the present disclosure are described herein. Reference to these examples is made in a non-limiting sense. They are provided to illustrate more broadly applicable embodiments of the present disclosure. Various changes may be made to the described embodiments, and equivalents may be substituted, without departing from the true spirit and scope of the present disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process acts or steps to the objective, spirit or scope of the present disclosure. Moreover, as will be understood by those skilled in the art, each of the individual variations described and illustrated herein has individual components and features that can be readily separated or combined with the features of some other embodiments without departing from the scope or spirit of the present disclosure. All such modifications are intended to be within the scope of the claims associated with this disclosure.

[0065]

[0073] Any of the above-described devices for performing a subject diagnostic or interventional procedure may be provided in packaged combination for use in performing such intervention. These supply "kits" may further include instructions for use and may be packaged in sterile trays or containers commonly used for such purposes.

[0066]

[0074] The present disclosure includes methods that may be performed using the subject devices. The methods may include the act of providing such a suitable device. This providing may be performed by an end user. In other words, the act of "providing" only requires the end user to obtain, access, approach, place, set up, activate, power on, or otherwise operate the device needed for the subject method. The methods recited herein may perform the recited events in any order that is logically possible, or may perform the recited order of events.

[0067]

[0075] Exemplary embodiments of the present disclosure are described above, along with details regarding material selection and manufacturing. Other details of the present disclosure will be understood in connection with the above-referenced patents and publications, as well as generally known or appreciated by those skilled in the art. The same may be true with respect to method-based embodiments of the present disclosure with respect to additional operations commonly or logically employed.

[0068]

[0076] Furthermore, while the present disclosure has been described with reference to several examples optionally incorporating various features, the present disclosure is not limited to what has been described or illustrated as contemplated with respect to each variation of the disclosure. Various modifications can be made to the described embodiments, and equivalents (whether stated herein or not included for purposes of brevity) can be substituted without departing from the true spirit and scope of the disclosure. Furthermore, when a range of values ​​is provided, it is understood that every intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the present disclosure.

[0069]

[0077] It is also contemplated that any feature of the described inventive variations may be presented and claimed independently or in combination with any one or more of the features described herein. Reference to a singular item includes the presence of a plurality of the same items. More specifically, as used in this specification and the claims relating thereto, the singular forms "a," "an," "said," and "the" include plural referents unless otherwise indicated. In other words, the use of these terms allows for "at least one" of the subject items in the above description and in the claims relating to this disclosure. Furthermore, it should be noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a predicate for the use of exclusive terms such as "solely," "only," etc. in connection with the recitation of claim elements or the use of a "negative" limitation.

[0070]

[0078] Without using such exclusive language, the term "comprising" in any claim related to this disclosure shall be deemed to permit the inclusion of additional elements or to transform the nature of the elements recited in such claim by the addition of functionality, regardless of whether a specified number of elements are recited in such claim. Except as specifically defined herein, all technical and scientific terms used herein should be given the broadest commonly understood meaning possible while maintaining the validity of the claims.

[0071]

[0079] The scope of the present disclosure should not be limited to the examples and / or specification provided, but rather should be limited only by the scope of the language of the claims appended to this disclosure.

Claims

1. In a 3D nanopore device for characterizing biopolymer molecules, a first Si layer deposited on the first dielectric layer; ３ N ４ Layer and; The first Si ３ N ４ a first metal layer deposited on the layer; a second dielectric layer deposited on the first metal layer; a second Si layer deposited on the second dielectric layer; ３ N ４ Layer and; The second Si ３ N ４ a second metal layer deposited on the layer; a third dielectric layer deposited on the second metal layer; a third Si layer deposited on the third dielectric layer; ３ N ４ Layer and; The third Si ３ N ４ a third metal layer deposited on the layer; a first plurality of elongated suppression electrodes etched and patterned in the second metal layer; a second plurality of elongated suppression electrodes etched and patterned in the third metal layer; the first and second plurality of parallel elongated suppression electrodes and the second and third Si ３ N ４ a plurality of nanopore channels etched in the second and third dielectric layers; each of the first plurality of elongated suppression electrodes is parallel to other elongated suppression electrodes of the first plurality of elongated suppression electrodes; each of the second plurality of elongated suppression electrodes is parallel to other elongated suppression electrodes of the second plurality of elongated suppression electrodes; the first and second pluralities of elongated suppression electrodes are orthogonal to one another; A 3D nanopore device characterized by:

2. 10. The 3D nanopore device of claim 1, further comprising a lower chamber fluidically connected to the plurality of nanopore channels.

3. The 3D nanopore device according to claim 1, further comprising: An upper chamber and a lower chamber, the first dielectric layer, the first Si ３ N ４ layer, the first metal layer, the second dielectric layer, the second Si ３ N ４ an upper chamber and a lower chamber, the upper chamber and the lower chamber being disposed between the upper chamber and the lower chamber; an electrolyte disposed in the upper chamber, the lower chamber, and the plurality of nanopore channels such that the upper chamber and the lower chamber are fluidly connected by the plurality of nanopore channels; A 3D nanopore device comprising:

4. 4. The 3D nanopore device of claim 3, further comprising upper and lower chamber electrodes disposed in the upper and lower chambers, respectively, wherein each of the upper and lower chamber electrodes is made of Ag / AgCl. ２ A 3D nanopore device comprising:

5. 4. The 3D nanopore device according to claim 3, wherein the electrolyte is KCl or LiCl. ２ A 3D nanopore device comprising:

6. 2. The 3D nanopore device of claim 1, wherein each of the plurality of nanopore channels has a diameter of about 2 nm to about 100 nm.

7. The 3D nanopore device according to claim 1, further comprising: a stack of dielectric layers including a second dielectric layer and a third dielectric layer; Second Si ３ N ４ layer and the third Si ３ N ４ layer, including Si ３ N ４ a stack of layers; a stack of metal layers including a second metal layer and a third metal layer; Dielectric layer: Si ３ N ４ layer, nanopore channels etched into each stack of metal layers and A 3D nanopore device comprising:

8. 2. The 3D nanopore device of claim 1, wherein the first plurality of elongated suppression electrodes and the second plurality of elongated suppression electrodes form an array that partially defines the plurality of nanopore channels.

9. 10. The 3D nanopore device of claim 1, further comprising a sensing electrode layer.

10. 10. The 3D nanopore device of claim 9, wherein the sensing electrode layer is configured to operate by ion blocking, tunneling, capacitive sensing, piezoelectric, or microwave sensing.

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