TUNNELING

This application claims priority to US Provisional application number 62/874,341 , filed July 15, 2019, the contents of which are hereby incorporated herein their entirety.

FIELD

[0001] Embodiments of the present invention are related to systems, methods, devices, and compositions of matter for identification and sequencing of biopolymers by electronic measurements. More specifically, this disclosure includes embodiments where electron tunneling nanogaps embedded in solid-state nanopores or nanoslits, which enable the detection of biopolymers electronically at a single unit or single-base level. The biopolymers are either linear, having a linear backbone, or having a linear carrier. BACKGROUND AND PRIOR ARTS OF THE INVENTION

[0002] With a state-of-the-art NGS sequencer, an individual human genome can be sequenced in a few days and around a thousand dollars. Compared to classical Sanger sequencing (-800 base read length on average), ¹ , however, NGS reads DNA much shorter. ² One of the disadvantages for the short reads is that it cannot encapsulate long blocks of repetitive sequences in the human genome, where half of the sequences are composed of repeats with the size from 1 - 2 bases to millions of bases. ³ That poses great challenges for the assembly of human genomes. Single-molecule real-time (SMRT) sequencing, known as the third-generation sequencing (developed by Pacific Biosciences), offers sequences with an average read length of > 10 kbp, ⁴ allowing for the de novo assembly of large genomes, such as a gorilla’s genome. ⁵ However, SMRT has a much higher error rate (~ 15%) for sequencing. A high sequencing coverage can overcome the error issue, but it will cost more, for example, with ~ $10,000 for a 30x human genome. For the use in clinics, ideally, the sequencing cost should be in a $100 per genome ballpark. ⁶

[0003] Nanopore sequencing is a technology based on the measurement of ionic current variations, which was conceptualized three decades ago. ⁷ A nanopore is an orifice with a diameter of nanometers, allowing the flow of ions across the nanopore under voltage bias. When a single-stranded DNA (ssDNA)— a polyanion— is

electrophoretically translocated through the nanopore embedded in membrane that separates two chambers filled with conductive electrolytes, it blocks an ionic current transiently. Since the nucleobases have distinguishable sizes, the blockage varies as the translocation proceeds. The DNA sequences can be deduced from the ionic current fluctuations. A commercial nanopore sequencer, Min ION, has been developed by Oxford Nanopore Technologies based on protein nanopores (www.nanoporetech.com). Since there is no theoretic limit on a length of DNA for the translocation, the nanopore sequencing overcomes the short read issues related to NGS. It may be an ultimate tool for de novo sequencing and analysis of structural variations. However, the protein nanopore sequencing is difficult to achieve single-base resolution. The overall sequencing accuracy is very low (85% with a single read ⁸ ). Gundlach and coworkers have demonstrated that the current blockage in a protein nanopore composed of mycobacterium smegmatis porin A (known as MspA) is a collected event of four nucleotides (quadromer), and therefore there are 4 ⁴ (i.e., 256) possible quadromers that exert a significant number of redundant current levels. ^{9 10} Because the ionic current is affected by nucleotides beyond those inside the nanopore, ¹¹ even an atomically thin nanopore may not be conceivable to achieve a single nucleotide resolution for DNA sequencing. [0004] Ventra et al. proposed to sequence DNA using a pair of electrodes separated by a distance of nanometers ¹² where electrons can tunnel through such a short gap, called nanogap. Since then, the work on sequencing by nanogap electron tunneling has much progress. ¹³ For the single-molecule sequencing by electron tunneling, one configuration is to embed a tunneling nanogap in a solid-state nanopore so the sequence of a single-stranded DNA can be read out sequentially by the nanogap when the molecule translocates through the nanopore. Given that the tunneling current is highly sensitive to changes in the gap size (~ an order of magnitude per A, comparable to the distance between two adjacent bases in a single stranded DNA), the tunneling measurement has the great potential to achieve a single nucleotide resolution for DNA sequencing. It has been demonstrated that nucleoside monophosphates and

oligonucleotides can generate tunneling currents in a small nanogap (< 1 nm). That poses a great challenge to manufacture such a small nanogap. The prior art has provided a method to fabricate sub-3 nanometer gaps composed of palladium electrodes (US 9,128,078). When the two electrodes are functionalizing with recognition molecules, the tunneling measurement was performed at a gap distance of ~ 2.5 nm. ¹⁴

SUMMARY OF THE INVENTION

[0005] The invention provides systems, devices, and methods for electronically sequencing biopolymers, such as DNA and RNA, proteins, sugars, etc., by electron tunneling with biopolymer movement control mechanism. This disclosure demonstrates the design, fabrication, and use of this type of device for DNA sequencing in a variety of exemplary embodiments. The same devices and methods can apply to the sequencing of proteins, peptides, polysaccharides, and other synthetic chemo-functional and biofunctional polymers. [0006] The present invention is an extension of previous applications (WO

2017/075620 and PCT/US18/32399). It uses the said devices and methods to control the motion of biopolymers in a nanopore for reading their sequences by an electron tunneling junction. The entirety of the two previous applications is included in this disclosure as a reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 shows a schematic illustration of motion-controlled nanopore DNA sequencing process by electron tunneling nanogap with recognition reader molecules under various embodiments: (a) a planar electron tunneling nanogap, (b) a stacked electron tunneling nanogap, (c) the process of DNA movement control and sequencing through a nanopore.

[0008] Figure 2 shows a schematic illustration of the process of fabricating a single stacked electron tunneling nanogap embedded in a nanopore, in which the two electrodes are in different planes separated by an insulating spacer. [0009] Figure 3 shows a schematic illustration of the process of fabricating double- stacked electron tunneling nanogaps embedded in a nanopore, in which two pairs of electrodes are involved for the tunneling measurements.

[0010] Figure 4 shows a schematic illustration of the process of fabricating a planar electron tunneling nanogaps embedded in a nanopore. [0011] Figure 5 shows a scheme for the synthesis of a xanthine-based reader molecule molecular models of xanthine interacting with DNA nucleosides based on molecular mechanics energy minimization [0012] Figure 6 shows molecular models of xanthine interacting with DNA

nucleosides calculated from molecular mechanics energy minimization.

[0013] Figure 7 shows a general form of structures for the xanthine-based reader molecules. [0014] Figure 8 shows a general form of structure of those with a smaller size derivated from the xanthine reader molecules.

[0015] Figure 9 shows a schematic illustration of the general structure of sample constructs.

[0016] Figure 10 shows a process of preparing DNA constructs.

DETAILED DESCRIPTION

[0017] An electron tunneling nanogap, composed of a pair of electrodes separated by a distance less than 3 nanometers, can be built in a nanopore either in a planar way (separated by a gap, see Fig. 1 a) or in a stacked way (separated by an insulating layer, see Fig. 1 b). DNA base sensing molecules (reader molecules) are attached to the electrodes such that the molecules on opposite electrodes do not touch each other but separated by a nanogap equivalent to the size of DNA bases. When a single-stranded DNA translocates through the nanopore, the individual nucleobases (117) can be captured by reader molecules attached to the electrodes to form a junction that facilitates the electron tunneling, creating electric signals for their recognition. The reader molecules interact with nucleobases via noncovalent bonds, such as hydrogen bonds, ¹⁵ which is relatively week in aqueous solution. Accurate base identification or sequencing of a biopolymer, such as DNA, requires the movement of the molecule through the nanopore to be slow (at millisecond level) and controlled (with sub nanometer precision). When a DNA molecule translocates through the nanopore without control, it is too fast (at microsecond level), so some individual nucleobases cannot be captured by the reader molecules, which results in deletion errors. Also, the fast movement of DNA does not give sufficient reaction time for the base-read molecule interaction to reach its equilibrium, which results in reading errors in the sequencing of nucleobases. Therefore, for the successful sequencing of a biopolymer using electron tunneling method, the slow and controlled movement of the biopolymer is an essential must-have condition. [0018] The present invention provides a system that has a mechanism to slow down and control the movement of the target biopolymer passing through a nanopore, enabling the sequencing of the biopolymer using electron tunneling. In general, the said system (Fig. 1 c) is consisted of an analysis stage equipped with a piezo actuator (100), a nanopore chip (140) with embedded tunneling nanogap (107) and reader molecules (110), a scan plate (130), a cis chamber (151 ) and a trans chamber (152), a DNA sample system (120) composed of a bead (108), a linker molecule (116) and the target DNA (109), and a voltage source for cross nanopore potential (115) and a voltage source for nanogap potential as well as signal measuring mechanism (111). The scan plate is placed substantially parallel to the nanopore chip. There are detailed

descriptions about the analysis stage design and composition, the scan plate design and fabrication, as well as DNA sample construction methods in the previous patent applications, WO2017/075620, and PCT/US18/32399, which are included here in their entirety. [0019] In some embodiments (ref. WO2017/075620), the biopolymer attaches to the scan plate directly through a chemical bond, either covalent or non-covalent, reversable or non-reversable, wherein the chemical bond is selected from the list comprising a biotin-streptavidin bond, an amide bond; a phosphodiester bond, ester bond, disulfide bond, imine bond, aldehyde bond, hydrogen bond, hydrophobic bonds, and a combination thereof.

[0020] In some embodiments, the system further comprises a controllable magnet, either an electromagnet or an adjustable magnet, or a group of magnet (ref.

WO2017/075620). In Fig.1 c (A-C), the bead is a magnetic bead, made of

paramagnetic, super-paramagnetic, ferromagnetic, or diamagnetic maerial. One end of the target single-stranded DNA molecule (ssDNA, 109) is attached to the magnetic bead (108) through a linker molecule (116) between them. The DNA sample with bead and linker molecule (120) is placed in the cis chamber (151). Under a biased potential through the voltage source 115, the free end of the ssDNA sample is pulled by electrophoretic force into a nanopore on the nanopore chip (140) and translocates through the nanopore into the trans chamber (152), while the other end is stopped at the nanopore entrance by the attached magnetic bead. By engaging or turning on the external magnet, the magnetic bead is attracted to the scan plate (130). By either applying strong magnetic force or through chemical bonding or other methods, the bead is tightly bound to the scan plate. Then, move the scan plate with the analysis stage (100) at a sub-nanometer precision (0.1 nm to 1 nm), and the DNA molecule will move through the nanopore at the same speed as the scan plate, and its bases can be read out by the reader molecules (110) one by one when they pass through the nanogap (107). For accurate base sensing by tunneling measurement, the base-reader interaction (residence) time is required to be 1 ms or longer. The longer the residence time, the more accurate the base sensing, however, the lower the sequencing throughput. So, generally, the preferred time is from 1 ms to 5ms per base unit, and less preferably 0.1 ms to 100 ms per base unit. For ssDNA, the base unit size is about 0.34 nm and spans 0.7 nm when fully stretched, which requires the scan plate to be moved with a speed of about 0.1 pm/sec to 1 pm/sec, less preferably 0.005 pm/sec to 10 pm/sec.

[0021] In some embodiments (ref. PCT/US18/32399), in order to achieve strong localized magnetic force to hold the magnetic bead tightly upto the scan plate, a layer of micro-soft magnetic structures (102) are contructed on the surface of the scan plate (101 ), either standout structures or patterned holes filled with permalloy. The soft magnetic structure has a layout of a solitary structure, a grid array, a hexagonal array, a solitary strip, a linear array of strips across an area, a patterned array of clusters of structures, a random pattern of structures, etc.. The soft magnetic structure may have the shape such as a circular cylinder, an oval cylinder, a rectangular block, a polygonal cylinder, a pyramid, an inverted pyramid, a cone, an inverted cone, an elongated shape, an irregular particle, a ring, etc. The size (diameter or width or equivalent dimension) ranges from 100 nm to 20 micrometer, preferably 1 micrometer to 5 micrometer. The center to center distance (or pitch) is usually 1 to 2 times the microstructure size.

Except a permalloy, other nickel and ion alloys can also be used as the core of the soft magnetic structure, such as a nickel-iron-molybdenum alloy, a nickel-cobalt alloy, an iron-nickel-cobalt alloy, an iron-silicon alloy, a nickel and iron alloy with different percentages of nickel and iron, any number between 0% and 100%.

[0022] In some embodiments, the scan plate has micro-pillars or patterned areas or an array of micro-pillars or patterned areas for the attachment of the target DNA molecules or other biopolymers. The array of micro-pillars or patterned areas are substantially aligned with the nanopore or nanoslit array on the nanochip (ref.

WO2017/075620).

[0023] In some embodiments, the target ssDNA molecule is attached to the scan plate by chemical bonding through a linker molecule without the bead. The target ssDNA molecule is ligated to the linker molecule and the linker molecule is attached to the scan plate. To identify or sequence the DNA, the scan plate is first lowered to allow the free end of the target ssDNA to enter the nanopore and translocate from the cis side to the trans side of the nanopore chip, and then move away from the nanopore. The target DNA can be sequenced when the ssDNA enters the nanopore and/or when it leaves the nanopore. It can be sequenced repeatly if needed to increase the accuracy.

[0024] In some embodiments, a double-stranded DNA or a single-stranded DNA, a polypeptide chain, a cellulose fiber or any flexible linear polymer, or the combination thereof, either natural, modified or synthesized, can used as the linker molecule (ref. WO2017/075620). A natural lambda DNA, which is about 48.5kb, 16.5 micrometer long

(double strand) or 34 micrometer long (single strand), is a good candidate for the linker molecule.

[0025] In some embodiment, a linker node, such as a non-magnetic bead or particle or a protein, is disposed between the linker molecule and the target DNA molecule and the linker node is configured to block the linker molecule from entering the nanopore to facilitate the alignment procedure (ref. WO2017/075620). The protein that can be used as a linker node includes but not limited to an antibody, an enzyme, a NeutrAvidin, a streptavidin, and an avidin. A linker node can be a polymer complex or particle or bead, a portion thereof, and a combination thereof. [0026] In another embodiment, the nanopore is a nanoslit with the dimension of width in the range of 1 to 50 nm, preferably 2 to 20 nm, most preferably 2 to 5 nm, and the dimension of length 5 nm to 1 pm or no greater than the bead size, preferably 10 to 500 nm, most preferably 20 to 100 nm. The planar nanogap is built across the width of the nanoslit.

[0027] In one embodiment, this invention provides a detailed process for the fabrication of an electron tunneling nanopore (Figs. 2A and 2B). A membrane layer (Fig. 2A, panel A, 202) is deposited on a base substrate (Fig. 2A, panel A, 201 ). The substrate 201 can be any material (such as Si-based, group III to V materials, or glass). Following the deposition, the substrate is etched from the backside to provide a supporting structure with a window (Fig. 2A, panel B, 203, and 204) for the tunneling nanopore device. A conductive layer (Fig. 2A, panel C, 205) is deposited as a bottom electrode on the 202 layer by a deposition technique including chemical vapor deposition (CVD), atomic layer deposition (ALD), physical vapor deposition (PVD), molecular vapor deposition (MVD), Electroplating, or Spin Coating etc., but not limited to those. The materials for the layer include but are not limited to metallic materials such as Au, Pt, Pd, W, Ti, Ta, Al, Ag, Cr, Cu, or conductive composite materials such as nitride compound (TiNx, TaNx) or oxide compound with or without doping. A various combination of sub-layers can be used as a multi-layer electrode to improve the adhesion and to control the conductivity of the electrode. In turn, an insulating layer (Fig. 2A, panel C, 206) is deposited by a technique of chemical vapor deposition (CVD), atomic layer deposition (ALD), physical vapor deposition (PVD), molecular vapor deposition (MVD), electroplating, or spin coating, etc., but not limited to those. The material can be any electrical insulator such as SiNx, SiOx, HfOx, AI2O3, and/or dielectrics used in the semiconductor industry. The insulating layer has a thickness of 2 to 5 nm, preferring to 3 to 4 nm. A 2 ^nd conductive layer (Fig. 2A, panel C, 207) is deposited as a top electrode by the same method as layer 205. A various combination of sub-layers can be used as a multi-layer electrode to improve the adhesion and control the conductivity of the electrode. A second electrically insulating layer (Fig. 2A, panel C, 208) is deposited on top of the conductive layer 207 as a cap layer to prevent the device from a short when exposed to an electrically conductive solution except for the exposed tunneling junction. A patterned etch mask is prepared to fabricate the tunneling junction embedded in nanopores using e-bam lithography on an e-beam resist or extreme ultraviolet lithography (EUV) on EUV resist, which serves as a etch mask after development. The resist mask is used as a pattern transfer (Fig. 2B, panel D, 209, 210) for a following multi-layered hard mask that serves as a final patterned etch mask. A reactive ion etching (RIE), plasma dry etching, focused ion beam (FIB), focused electron beam (FEB), or ion beam etching (IBE) is performed to etch through the cap layer (208), top electrode (207), insulating layer (206), bottom electrode (205), and the membrane (202). A stacked tunneling nanogap embedded in a nanopore (Fig. 2B, panel E, 211 ) is created after removing the etch residues and the etch mask. The nanopore is sized with a diameter of 1 to 50 nm, preferably 2 to 20 nm, most preferably 2 to 5 nm.

[0028] In another embodiment, this invention provides a process to fabricate a device with two tunneling gaps (four electrodes) with the same type or different types of reader molecules embedded in a nanopore (Fig. 3A and B) for reading polymeric sequences repeatedly, which increases the sequencing accuracy. The tunneling gaps are separated by a spacer (Fig.3A, panel C, 308). The spacer can be any electrically insulating material such as SiNx, SiOx, HfOx, AI2O3, and/or dielectric materials used in the semiconductor industry. The deposition of the spacer is performed with a technique of chemical vapor deposition (CVD), atomic layer deposition (ALD), physical vapor deposition (PVD), molecular vapor deposition (MVD), electroplating, or spin coating, etc., but not limited to those. The other process for the fabrication of the device follows those described in [0027] In considering a higher aspect ratio of the tunneling junction compared to a single tunneling nanogap, a multi-layer hard mask approach is preferable, using a patterned resist as a pattern transfer mask, as mentioned in [0027]

[0029] In one embodiment, this invention provides a detailed process for the fabrication of a device composed of a planar tunneling nanogap embedded in a nanopore (Fig. 4A and B). An insulating layer (Fig. 4, panel A, 402) is deposited on a base substrate (Fig. 4, panel A, 401 ). The substrate 401 can be any material (such as Si-based, group III to V, or glass). Following the deposition, the substrate is etched from the backside to provide a supporting structure with an open window (Fig. 4, panel A, 403, and 404) for the tunneling nanopore device. In turn, an electrically conductive layer (Fig. 4, panel A, 405) is deposited on the insulating layer 402. The conductive layer can be a multi-layer structure to enhance the adhesion and to control the desired

conductivity. An adhesive layer is deposited to either act as a second conductive electrode or insulating/protective layer (Fig. 4, panel A, 409) if needed. A reactive ion etching (RIE), plasma dry etching, focused ion beam (FIB), focused electron beam (FEB), or ion beam etching (IBE) is performed to fabricate a nanowire. After etching and removing of the resist residue and cleaning the substrate, an electrically insulating materials (Fig. 4, panel A, 406) is deposited as a capping layer to protect the electrode when exposing in a conductive solution except for the nanogap cross-section. A non- conductive layer (Fig. 4, panel A, 410) as an adhesion promoter is deposited for the photoresist if needed. Photoresist (Fig. 4, panel A, 407) can be patterned using electron beam lithography (EBL) or extreme ultraviolet lithography (EUV) to define the nanogap cross the nanowire (Fig. 4, panel A, 408). This patterned resist can act as a direct etch mask or can be used for the pattern transfer to fabricate any hard mask. The gap pattern is crossing the nanowire pattern. The top of the sacrificial layer (410) or the cap insulating layer (406) can be seen between the gap. A reactive ion etching (RIE), plasma dry etching, focused ion beam (FIB), focused electron beam (FEB), or ion beam etching (IBE) is performed to etch through the layers to form a nanopore with an embedded tunneling nanogap as shown in Fig. 4B. After strip/cleaning, the topmost layer can be the sacrificial layer (410) or the cap insulating layer (406).

[0030] In some embodiments, the reader molecules are attached to those electrodes that form a tunneling nanogap to interact with an individual base unit of a polymer for their identification. The said interaction is hydrogen bonding, stacking, electrostatic, or other noncovalent interactions.

[0031] In some embodiments, the reader molecules disclosed in the prior arts, including 1 .8- Napthyridine derivatives and imidazole-carboxamide derivatives (US 8,628,649), benzamide (US 9,140,682), triazole-carboxamide derivatives (US

10,336,713), benzimidazole-2-carboxamide (US 2016/0108002), pyrene derivatives (US

2019 / 0195856), are used to read the basic units of bio- and synthetic polymers by electron tunneling.

[0032] In one embodiment, this invention exploits xanthine as a reader molecule (Figure 5, 503) for reading sequences of biopolymers including naturally occurring nucleic acids, proteins, peptides, and polysaccharides as well as those synthetic nucleic acids such as XNA and nucleic acids analogs such as peptide nucleic acid (PNA), re engineered proteins with unnatural amino acids, modified peptides. The compound 503 is synthesized following a route, as shown in Fig. 5, starting from 8-bromoxanthine (501 ). [0033] In one embodiment, molecular modeling indicates that the reader molecule 503 interacts with DNA bases through hydrogen bonding to form different triplet complexes (Fig. 6) with two sulfur atoms fixed at a distance of 2.8 nm so the tunneling currents would flow through these structures differently, which are used as signatures for individual nucleosides. When a DNA molecule translocates through the tunneling nanogap, its sequence can be read out by the tunneling signatures. Given that the Au-S bond has a length of 2.156 A, ¹⁶ a nanogap with a size of ~ 3.2 nm is practical for the tunneling sequencing. That provides a manufacturing advantage for the fabrication of the tunneling nanopore devices compared to those tunneling junctions with their gap sizes around 2.5 nm.

[0034] In some embodiments, the structure of reader molecules can be described as a general form, as expressed in Fig. 7. It is composed of a recognition moiety that interacts with the monomers of a polymer through noncovalent forces and an anchor that is used to fix the recognition moiety on electrodes, both of which are connected through a linker 701 , 702, or 703, which allows different electron tunnelings.

[0035] In some embodiments, the invention provides a series of reader molecules with a smaller size derived from the xanthine reader molecules (Fig. 8). These reader molecules are suitable to the tunneling nanogaps, preferably with their sizes around 2.5 nm. Their linkers 801 , 802, or 803 are equivalent to those corresponding 701 , 702, or 703, respectively.

[0036] In some embodiments, this invention provides a method to prepare a biopolymer sample construct for its analysis by the said tunneling nanopore device. As shown in Figure 9, the construct has a polymeric target (Fig. 9, 903) attached to a magnetic bead (901 ) through a molecular linker (902), tailed with an oligonucleotide to prevent the target from jumping out of the nanopore. The size of magnetic beads ranges from 50 nm to 20 pm, preferably 1 pm to 3 pm, and the molecular linker comprises negatively charged DNA (either single-stranded or double-stranded) and RNA, neutral polyethylene glycol (PEG), or positively charged polyethyleneimine under the physiological conditions, but not limited to them. The polymeric target is naturally occurring DNA, RNA, proteins, polysaccharides, and their modified artificial

counterparts. The oligo tail is composed of negatively charged DNA (either single- stranded or double-stranded) and RNA, neutral polyethylene glycol (PEG), or positively charged polyethyleneimine under the physiological conditions, but not limited to them. [0037] This invention provides examples for the preparation of DNA constructs. One example (delineated in Fig. 9b) is a DNA construct. First, l-DNA functionalized with an amine at its one end, used as a linker molecule, is attached to a magnetic bead functionalized with carboxylate via an amidation reaction (Step A). ¹⁷ In other

embodiments, this step employs different chemical reactions such as azide-alkyne cycloaddition, maleimide-thiol coupling, etc. In parallel, a DNA target is ligated to a linear M13mp18 DNA tail via a T4 DNA ligase (Step B). ¹⁸ In some embodiments, this step is finished via a chemical reaction or other enzymatic ligation such as T7, Taq, etc. Then, the target-tail conjugate is linked to the l-DNA linker attached to the magnetic bead through the T4 DNA ligation (Step C). In some embodiments, this step is completed by another ligase such as T7, Taq, etc., or chemical reactions such as, but not limited to amine-carboxylate, thiol-maleimide, or click coupling. ^{19 20} In some embodiments, the sample construct is prepared firstly by connecting a linker molecule, a target molecule, and a linker molecule to form a linker-target-tail conjugate that is then attached to a magnetic bead. [0038] Another example is preparing a DNA construct starting with a double- stranded DNA sample, using the linear pUC19 vector as a tail (Fig. 10). First, l-DNA is attached to magnetic beads via an azide-alkyne click reaction (Step A). In parallel, the double-stranded DNA target is ligated to a double-stranded DNA tail through the T4 DNA ligation (Step B). In some embodiments, different ligases such as T3, T7, Taq are be used depending on targets to be ligated. The target-tail conjugate is then conjugated to the l-DNA on the magnetic bead (Step C). In turn, the double-stranded DNA construct on the magnetic bead is digested by l-exonuclease ²¹ to obtain a single- stranded DNA construct on the magnetic bead (Step D). In another embodiment, the ligated double-stranded linker-target-tail complex is prepared and digested before the attachment to the magnetic bead.

[0037] In some embodiments, a nanochip containing an array of nanopores between 100 to 100 million, preferably between 1 ,000 to 1 million, is made in order to satisfy the throughput requirements of biopolymer sensing or sequencing

[0038] In some embodiments, an array of nanopore devices on one chip is divided into multiple regions or modules and the signals are read out separately from one region to other regions by separate signal recording units in order to overcome the bandwidth and sampling frequency limits of a single recording unit.

GENERAL REMARKS

[0039] All publications, patents, and other documents mentioned herein are

incorporated by reference in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. While the present invention has been illustrated by a description of various embodiments and while these embodiments have been described in considerable detail, it is not the intention of the applicants to restrict or in any way limit the scope of the applications. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details,

representative device, apparatus and method, and illustrative example shown and described. Accordingly, departures may be made from such details without departing from the spirit of the applicant’s general inventive concept.

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