Biopolymer Sequencing System

Abstract

The present invention provides systems and related devices, methods and molecular modules for electronically sequencing biopolymers.

Description

[0001] Priority claim

[0002] This application claims priority to U.S. Provisional Application No. 62 / 772,837, filed on November 29, 2018, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] Embodiments of the present invention relate to systems, methods, devices, and compositions of matter for identifying and sequencing biopolymers using electronic measurements. More specifically, the present disclosure includes embodiments that design, synthesize, and functionalize molecular clefts or tweezers with molecular wires having binding functions that bridge nanogaps, enabling one to electronically identify biopolymers based on their components at the single molecule level. Background of the Invention

[0005] Next-generation sequencing (NGS) has reduced the cost and time of sequencing an individual human genome. However, the DNA sequences read by NGS are 2 Much shorter than classic Sanger sequencing (average read length of ~800 bases). 3 One of the drawbacks is that short reads cannot encapsulate long blocks of repetitive sequences, resulting in a fragmented assembly of the human genome, with half of the sequence consisting of repeats ranging in size from 1 base (mononucleotide repeats or homopolymers) or 2 bases (dinucleotide repeats) to millions of bases, and in copy numbers ranging from two to millions. 4 Single-molecule real-time (SMRT) sequencing—also known as third-generation sequencing (developed by Pacific Biosciences)—provides sequences with an average length of >10 kbp 5 , allowing the de novo assembly of large genomes, such as that of the gorilla. 6 However, SMRT sequencing has a much higher error rate (about 15%). Although errors can be overcome with high sequencing coverage (>30x), it costs about $10,000 for a 30x human genome. For clinical use, the cost of sequencing each genome should ideally be $100. 7

[0006] Nanopore sequencing is a technology based on measuring ionic currents that was conceptualized 30 years ago. 8A nanopore is an orifice with a diameter of nanometers that allows a fluid to conduct ions under a bias. When a single-stranded DNA—a polyanionic molecule—is electrophoretically transferred through a nanopore embedded in a membrane separating two chambers filled with a conducting electrolyte, it causes a transient blockage of the ionic current. Since the nucleobases have distinguishable sizes, the blockage changes as the translocation proceeds. The DNA sequence can therefore be deduced from the fluctuations in the ionic current. The MinION sequencer ( www.nanoporetech.com ). Since there is no theoretical limit to the length of translocated DNA, nanopore sequencing may be the ultimate tool for de novo sequencing and structural variation analysis, benefiting from long read lengths, which overcomes the problems associated with short read lengths of NGS in genome assembly. However, protein nanopores can only achieve low sequencing accuracy (85% for a single read). 9 Gundlach and colleagues have shown that current blockade in a protein nanopore composed of the Mycobacterium smegmatis porin A (called MspA) is an aggregate event of four nucleotides (tetramers), so there are 4 4 (ie, 256) possible tetramers yield a large number of redundant current levels. 10，11 Since the ionic current is affected by nucleotides other than those in the nanopore, 12 The concept of atomically thin nanopores for sequencing may not be able to achieve single-nucleotide resolution.

[0007] Ventra et al. proposed using a pair of electrodes separated by a nanometer distance 13 To sequence DNA, electrons can tunnel through such a nanogap. Since then, great progress has been made in reading nucleobases via electron tunneling. 14 With the tunneling nanogap embedded in a solid-state nanopore, single-stranded DNA can be sequenced by sequentially shifting its nucleotides across the gap. Tunneling measurement provides a highly spatially resolved method for DNA sequencing and can achieve single-nucleotide resolution because the distance between two adjacent bases in single-stranded DNA is greater than However, it requires a sharp electrode to achieve single-base resolution. It has been demonstrated that monophosphate nucleosides and oligonucleotides can generate tunneling currents across small nanogaps (<1nm), but the signals overlap in naturally occurring DNA nucleotides. 15，16 Verified by theoretical calculations. 17，18 Although tunneling measurements were performed at a gap distance of about 2.5 nm by functionalizing the two electrodes with recognition molecules, 19 However, it is currently technically challenging to fabricate tunneling gaps <3 nm.

[0008] Collins and colleagues created a single-walled carbon nanotube (WCNT) field-effect transistor (FET) device to which the Klenow fragment of DNA polymerase I was tethered to monitor its DNA synthesis. 20，21 In this setup, a brief shift of ΔI(t) below the average baseline current is recorded when a nucleotide is incorporated into a DNA strand. Enzyme incorporation of different nucleotides results in differences in ΔI. This technique has the potential to be used for DNA sequencing. A carbon nanotube is a material made of only a single layer of carbon atoms locked in a hexagonal grid. It is a rigid chemical structure. Its sensing may rely on electrostatically gated motion of charged side chains close to the protein attachment site.

[0009] It has been reported that silicon nanowires constructed on the edge of SiN nanopores can detect DNA translocation by sensing potential changes. 22 In addition, field-effect transistors (FETs) can sense conformational changes near the gating conductance of semiconductor channels in physiological buffers, allowing highly sensitive detection of ligand-receptor interactions. 23 However, these FET devices have not demonstrated the ability to read individual DNA bases in a DNA strand.

[0010] A nanojunction can be formed by connecting a molecular wire to two electrodes separated by a nanoscale gap. When integrated into a circuit, it allows electrons to flow. Typically, the molecular components are covalently attached to the electrodes, and the electronic conduction of the junction is affected by the molecular structure and the molecule-metal contact. 24 However, its electronic states can be switched via stereoelectronic effects 25 , and can be altered by external stimuli. For example, the conductance of a host-guest molecule junction can be modulated by the insertion of a guest molecule. 26 Furthermore, protein transistors can be fabricated by bridging the nanogap using anti-gold nanoparticle antibodies. 27

[0011] Electron transfer (ET) can be mediated with proteins and peptides. 28，29 It can be said that ET through peptides can operate in parallel via tunneling and hopping; however, their contributions vary with the length of the intervening bridge. For short bridges, tunneling dominates, while for long bridges, hopping becomes more pronounced. 30 Isied and colleagues demonstrated this experimentally. 31 Side chain composition, hydrogen bonding, and alpha-helical secondary structure have been identified as important factors affecting short-range hopping and tunneling conductivity in these peptide systems. Therefore, it is possible to tune the charge transfer properties of peptides by manipulating their secondary structure.

[0012] The present invention provides a system equipped with nanodevices to sequence biopolymers. One of these is a nanogap composed of nanoelectrodes. For sequencing biopolymers, the nanogap is bridged by a wire entity that can capture individual monomers in the polymer chain by forming non-covalent complexes, resulting in changes in the structure and subsequent changes in the wire conductance. Since each complex is different from the other, it has a different effect on the conductance. The current fluctuations can be used to identify individual monomers in the polymer sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of a molecular tweezers-nanopore system for sequencing DNA according to various embodiments is shown.

[0014] Figure 2 A schematic diagram of a system showing paired electrodes in a planar configuration.

[0015] FIG3 shows the chemical structure of the molecular cleft (a) and its DFT (density functional theory) model (b).

[0016] Figure 4 Shown are the DFT structures of the cleft complexes with DNA nucleotides and their binding energies.

[0017] Figure 5 The molecular cleft is shown in complex with nucleobases in a single-stranded DNA fragment.

[0018] Scheme 1 shows the route to synthesize the molecular cleft (MC-1) bearing an acetylene group.

[0019] Figure 6 Peptide lines with alpha helical structure are shown.

[0020] Figure 7 The three-dimensional structure of the molecular cleft-peptide conjugate calculated by molecular mechanics is shown.

[0021] FIG8 shows the chemical structure of the molecular tweezers (a) and its DFT model (b).

[0022] Scheme 2 shows the route for the synthesis of molecular tweezers (MT-1) bearing an acetylene group.

[0023] FIG. 9 shows the DFT structure of the complex of the molecular tweezers and hydrogen bonding part with DNA nucleotides and the binding energy calculated by DFT calculation.

[0024] FIG. 10 shows the three-dimensional structure of the molecular tweezers-peptide conjugate (a) and its complex with the nucleobase in DNA (b).

[0025] Scheme 3 shows the route to synthesize the molecular tweezers (MT-2) with two acetylene groups.

[0026] Fig.11 The alpha helical structure of a short peptide strand with a high percentage of aromatic amino acid residues is shown.

[0027] Fig.12 The structure of the molecular tweezers-peptide-wire hybrid is shown.

[0028] FIG. 13 shows the structures of a conjugate of a peptide wire having a hydrogen bonding moiety (a) and a hybrid incorporated into the hydrogen bonding moiety of the peptide (b).

[0029] FIG. 14 shows the chemical structures of reagents used to functionalize the ends of peptide wires to allow their attachment to metal electrodes.

[0030] Scheme 4 shows a route for the synthesis of a tripod anchor for attaching peptides to metal surfaces.

[0031] Fig.15 The chemical process of connecting peptide wires to metal electrodes functionalized with tripod molecules is shown.

[0032] Fig.16 Shown is the process of connecting a peptide wire to two electrodes composed of different materials that make up the nanogap. SUMMARY OF THE INVENTION

[0034] The present invention provides a system comprising a nanojunction that can sense chemical entities for electronically sequencing biopolymers (e.g., DNA and RNA), a nanopore (or nanochannel) for transmitting a single biopolymer to a sensing device, the movement of which is controlled by a mechanical device. The present invention provides a molecular probe connected to a molecular wire for the identification of a single chemical entity in a sample molecule. The same system, device, and method are applicable to sequencing proteins, peptides, polysaccharides, etc. Here, the present disclosure shows the design, manufacture, and use of such a device for sequencing DNA.

[0035] In one embodiment of the present invention ( Figure 1), the system comprises: (a) a nanopore embedded in a membrane separating two fluid reservoirs, allowing the biopolymer to be translocated from one side to the other; preferably the diameter of the nanopore is 2nm to 1μm; more preferably the diameter is 3nm to 40nm, and most preferably 3nm to 10nm; (b) a pair of stacked electrodes separated by a dielectric layer, which are located inside the nanopore; preferably the thickness of the dielectric layer is 3nm to 100nm, more preferably the thickness is 4nm to 40nm, and most preferably the thickness is 4nm to 10nm; the electrode pairs are made of the same material or different materials. Moreover, multiple electrode pairs are established in the nanopore, so when the biopolymer passes through the nanopore device, it will be sequenced multiple times. (c) A linear molecular module that can interact with a single component of the biopolymer through non-covalent binding, which has two anchoring sites for covalent binding to paired electrodes; under bias, the molecular module allows electrons to be transferred from one electrode to the other electrode; (d) a mechanism to control the translocation speed of the polymer molecules through the nanopore to improve the measurement accuracy. The motion control mechanism can be an enzyme (such as a polymerase or helicase) combined with a viscous medium and / or some electrophoretic force, or a nano-precision piezoelectric drive or any electromechanical drive and can be controlled by an adjustable magnet, or a combination of all of the above. The embodiment shows that when the single-stranded DNA is displaced through the nanopore, its movement is controlled using a piezoelectric drive with sub-nanometer resolution described in our previous invention (described in PCTWO2017075620A1). For sequencing DNA, the preferred speed is 5ms (milliseconds) to 20ms per base; the second preferred speed is 1ms to 100ms per base, and the less preferred speed is 0.1ms to 1000ms per base.

[0036] In another embodiment, the paired electrodes are not stacked in the nanopore, but are fabricated at the trans or cis outlet of the nanopore, on the top surface of the nanopore ( Figure 2 ). Thus, the resulting planar nanogap between the two electrodes is formed above the center of the nanopore, off-center, or along the edge of the nanopore. The preferred diameter of the nanogap is 2nm to 40nm, and the most preferred diameter is 4nm to 10nm. The planar nanogap can be electrochemically manufactured by feedback control of the nanopore. 32，33

[0037] In another embodiment of the present invention, paired electrodes are laterally manufactured in nanochannels and functionalized by the molecular modules. Polymer molecules such as DNA flow into nanochannels and then pass through nanogap, where their sequences are read out. DNA motion is controlled by enzymes (such as polymerases or helicases) combined with viscous media and / or some electrophoretic forces, or by precise piezoelectric actuators or precise electromechanical actuators, by adjustable magnets, by electrophoretic forces combined with viscous media, or by micro-features in microchannels such as micro-pillars (pillars), porous media, etc., stretching DNA and slowing down its movement speed.

[0038] In some embodiments of the invention, the biopolymer (e.g., DNA) is attached to a bead so that when it passes through the gap, it can stop at the entrance of a nanopore or nanochannel near the nanogap and then be pulled away from the nanogap in a precisely controlled manner so that its components can be identified by the wire molecular probe as it passes through the gap.

[0039] In some embodiments of the present invention, a molecular cleft is designed and synthesized as a molecular probe to recognize nucleobases for sequencing nucleic acids, which consists of two aromatic motifs attached to a pyridine platform ( FIG. 3 ).

[0040] In one embodiment, the molecular clefts form complexes with DNA nucleobases individually, trapping them in the hollow space through hydrogen bonding and stacking interactions. These complexes have different structures and different binding energies, which can be used as features to identify individual nucleobases ( Figure 4 ).

[0041] In another embodiment, the molecular cleft can be formed by trapping single-stranded DNA in the hollow space, forming a complex with a single nucleobase in the single-stranded DNA ( Figure 5 ).

[0042] One embodiment of the present invention provides a method for synthesizing the molecular cleft (MC-1) comprising an acetylene functional group for its coupling with a molecular wire (Scheme 1).

[0043] In some embodiments, peptides are used as conductive molecular wires to bridge electrodes. Therefore, the peptide molecular junction coupled to the molecular cleft is used as a sensing component. When a nucleobase binds to the molecular cleft, it disturbs the conformation of the peptide molecule, which in turn causes a change in its conductance. Each nucleobase has a different effect on the conductance of the peptide wire, so we can identify them by measuring the current.

[0044] In one embodiment, the molecular wire is a peptide having an alpha helical structure having the sequence NH2-ELKAIAQEFKAIAKEFKAIAFEFKAIAKQK, comprising aromatic and charged amino acid residues ( Figure 6 ). The peptide wire is conductive. 34

[0045] In one embodiment, the present invention provides a conjugate of a peptide line and a molecular cleft, which is shown as a T-shaped molecule. First, the peptide is modified, wherein one phenylalanine is replaced by azidophenylalanine. Thus, a molecular cleft such as MC-1 is reacted with the modified peptide in the presence of a copper catalyst to form a molecular module, wherein one peptide is coupled to one molecular cleft ( Figure 7 ). 35

[0046] In some embodiments, the molecular cleft is composed of two different aromatic motifs, one for π-π interactions and the other for forming hydrogen bonds with the nucleobases attached to the five-membered aromatic ring, referred to as molecular tweezers.

[0047] In one embodiment, the molecular tweezers are molecules composed of a hydrogen bonding moiety, a triazole carboxamide, and an aromatic stacking moiety anthracene connected by a pyrrole structure ( FIG. 8 a ). The DFT model shows that the molecule has a tweezer shape ( FIG. 8 b ).

[0048] In one embodiment, the present invention provides a method for synthesizing a molecular tweezer comprising an acetylene functional group for forming a conjugate with the peptide wire (Scheme 2, MT-1).

[0049] The molecular tweezers are closed when they interact with the nucleobase. DFT calculations show that it forms a complex with the nucleobase through horizontal hydrogen bonding and vertical π-π stacking (Figure 9). The interaction of the molecular tweezers and the nucleobase forms non-covalent complexes with different structures, resulting in distinguishable binding energies. These features can be used to identify the nucleobase. In contrast, the complex formed by the molecular tweezers with the DNA nucleotides is more stable than the hydrogen bonding part alone.

[0050] The molecular tweezers form a conjugate with the peptide wire through a click reaction ( FIG. 10 a ). The conjugate interacts with a single nucleobase in the single-stranded DNA through the molecular tweezers ( FIG. 10 b ).

[0051] In another embodiment, the present invention provides a method for synthesizing a molecular tweezer with two acetylene functional groups (Scheme 3, MT-2), so that it can be incorporated into a peptide wire via a triazole isostere forming an amide bond.

[0052] In some embodiments of the invention, the peptide strand is a short sequence with a high percentage of aromatic amino acid residues: tyr-tyr-arg-lys-tyr-lys-glu-tyr-asp-asp-tyr( Fig.11 ). 36

[0053] Two short peptides were coupled to the molecular tweezers via the triazole moiety to form a hybrid of two peptides and a molecular tweezer, which appeared as a Y-shaped molecule ( Fig.12 ), where the molecular tweezers act as molecular switches. When interacting with the nucleobases, it changes the current flowing through the peptide wire by regulating the conformation of the peptide wire.

[0054] In one embodiment of the invention, it provides a conjugate of a peptide wire with a hydrogen bonding moiety that interacts with a nucleobase via hydrogen bonding ( FIG. 13 a ), and also provides a hybrid of a hydrogen bonding moiety with a peptide ( FIG. 13 b ). Compared to molecular tweezers, the hydrogen bonding moiety interacts less strongly with a DNA nucleobase.

[0055] Some embodiments of the present invention provide methods and reagents for connecting peptide wires, their conjugates, and their hybrids to electrodes. In one embodiment, the present invention provides reagents cysteine ​​(20) or (S)-2-amino-3-(1,2-dithiolan-4-yl)propionic acid (21), which are described in the literature 37 The peptides were synthesized according to the method reported in for N- and C-terminal functionalization of peptide threads (Figure 14). These reagents were incorporated into the peptide threads by conventional peptide synthesis.

[0056] In some embodiments, the present invention provides a tripod anchor for connecting peptide wires, their conjugates, and their hybrids to metal electrodes. Since the Au-S bond is only a semi-covalent bond, the monothiol-anchored monolayer is unstable enough to be easily displaced from the surface by other thiols. It can be desorbed from the surface within a few days when stored in phosphate buffer and exposed to high temperature and high salt concentration. The trisulfide anchor will overcome these limitations of the single sulfur anchor. 38 .

[0057] In one embodiment, the present invention provides a method for synthesizing a tripod anchor (Scheme 4) with a formylphenylboronic acid functional group for anchoring peptide wires. Its adamantyl group makes the anchor more rigid than an alkyl chain. The tripod provides a firm contact with the metal surface, making the measurement of single-molecule junction conductance reliable. 39 To connect the peptide threads, they were functionalized at the termini with 3,4-dihydroxyphenylalanine (DOPA). In the presence of N-tert-butylhydroxylamine hydrochloride, DOPA reacts specifically with boronic acid to form a stable molecular junction ( Fig.15 ). 40

[0058] In one embodiment, the present invention provides a method of connecting peptide wires to electrodes composed of different materials that make up a nanogap. Fig.16 An example of a nanogap containing a metal electrode and a graphene electrode is shown. For connection, the metal electrode is functionalized with a tripod anchor and the graphene is functionalized with a p-(N-maleimido)phenyl functional group. At the same time, one end of the peptide wire is modified with DOPA and the other end is modified with cysteine. 41 Therefore, the peptide wire is connected to the two electrodes through two different reactions. DETAILED DESCRIPTION

[0059] Computer modeling was performed in the software Spartan'18 (Wavefunction Inc.). The 2D molecular structures were built in ChemDraw pro v17 and then converted to 3D structures in Spartan'18. Each monomer structure was first energy minimized using the built-in Merck molecular force field (MMFF) and then calculated using B3LYP / 6-31G* in a water environment. The complexes were built with the help of MMFF and B3LYP / 6-31G* were calculated in a water environment in Spartan'18. The bond energies of the complexes were calculated by subtracting the energies of the individual monomers from the total energy of the complex.

[0060] Scheme 1 shows the method for synthesizing a molecular cleft (MC-1) with an acetylene group. The synthesis starts with 4-(trimethylsilyl)ethynylpyridine-2,6-dicarboxylic acid (1) 42 Reaction with 2-naphthylmethylamine (2) in the presence of DCC affords product 3 containing a trimethylsilyl-protected ethynyl functional group. The protecting group can be obtained by using n Compound 3 was removed by treatment with Bu4NF, providing the final product MC-1 that can be connected to the molecular wire.

[0061] Scheme 2 shows another method for synthesizing a molecular cleft with an acetylene group (MT-1). The synthesis starts with the reaction of dimethyl 1H-pyrrole-3,4-dicarboxylate (4) with bromoethynyltriisopropylsilane, and the copper complex 43 Catalysis affords a product (5) containing an ethynyl functional group protected by triisopropylsilyl (TIPS). Compound 5 is converted into a dicarboxylic acid (6) by hydrolysis under alkaline conditions. Subsequently, compound 6 is reacted with 2-(aminomethyl)anthracene and 5-(aminomethyl)-1,2,4-triazole-3-carboxamide in the presence of DCC to afford a molecular cleavage product (8) containing an ethynyl functional group protected by triisopropylsilyl (TIPS). The desired product (MC-2) is obtained by removing the TIPS protecting group with tetra-n-butylammonium fluoride (TBAF).

[0062] Scheme 4 shows a method for synthesizing an anchor molecule (A-1) having a trivalent thiol functional group. The starting material 1-(4-bromophenyl)-3,5,7-tris(trifluoromethanesulfonylmethyl)adamantane (14) was synthesized according to a method reported in the literature. 44 First, it was formylated by reaction with dichloromethyl methyl ether (Cl2CHOMe) in the presence of silver trifluoromethanesulfonate (AgOTf) to provide the aldehyde 15, which was then sequentially converted to the acetal 16. 45 Compound 16 is then treated with potassium thioacetate to afford the thiolated compound (17). Compound 17 is converted to the boronic acid 18 by photoinduced borylation.46 The desired compound 19 was obtained by deacetylation with an acid. During the functionalization of the electrode, the acetyl group was removed in situ using pyrrolidine.

[0063] More specifically, the present invention includes the following contents:

[0064] 1) A device for direct electronic identification and / or sequencing of biopolymers, comprising:

[0065] a. A silicon chip disposed between a cis chamber and a trans chamber, wherein the nanopore is embedded in an effective thickness that can accommodate at least a portion of a polymer molecule when the polymer molecule is translocated from the cis chamber to the trans chamber;

[0066] b. a first electrode and a second electrode embedded in the nanopore and overlapping each other, which are separated by a dielectric layer to form a nanogap therebetween;

[0067] c. Synthetic T-shaped or Y-shaped modular modules that can act as a bridge between two electrodes, allowing current to flow from one end to the other; and receptors that can capture biopolymers by interacting with their components. The interaction causes conformational changes in the receptor as well as the rest of the molecule, which changes the current flowing through one electrode to the other, thus allowing recognition of the biopolymer or its monomers;

[0068] Among them, the silicon chip can have a single nanopore or multiple nanopores; the materials of the electrodes can be the same or different, such as metals such as gold, silver, palladium, platinum or metal alloys, conductive non-metals such as graphene, or two different metals or two different conductive non-metals. Different materials allow the molecular bridge to be connected to the electrodes in a controlled manner. The thickness of the dielectric layer between the two electrodes is 3 to 40nm, preferably 4 to 10nm. The bridging molecule can be synthesized into one molecule, or it can be constructed by coupling molecular tweezers (cleft receptor structure) with molecular wires (linear structure).

[0069] 2) Device for direct electronic identification and / or sequencing of biopolymers, comprising:

[0070] The same components as the above device, except that the electrode pair is fabricated on the same plane of the nanopore surface and separated by a nanogap at a distance of 2 to 100 nm, preferably 3 to 10 nm, whether on the inlet side or the outlet side; wherein the nanogap is located at the center of the nanopore, off-center, or along the edge of the nanopore.

[0071] 3) Device for direct electronic identification and / or sequencing of biopolymers, comprising:

[0072] a. A nanochannel with a gradually decreasing channel size, wherein the nanochannel continuously and smoothly or stepwise decreases its size and has or does not have microstructures on the floor or walls that help stretch and slow down the biopolymer;

[0073] b. A first electrode and a second electrode are placed at the ends of a nanochannel, wherein the first electrode is embedded in one side of the nanochannel and the second electrode is embedded in the other side of the nanochannel, facing each other and forming a nanogap therebetween; or, the two electrodes are embedded in the same side of the nanochannel with a dielectric layer therebetween, similar to the arrangement in the nanopore device described in 1), but are always located at the ends of the nanochannel; similar to device 1), the electrodes are made of the same or different materials.

[0074] c. Synthetic T-shaped or Y-shaped modular modules that can act as a bridge between two electrodes, allowing current to flow from one end to the other; and receptors that can capture biopolymers by interacting with their components. The interaction causes conformational changes in the receptors as well as the rest of the molecule, thus changing the current flowing from one electrode to the other, so that the biopolymers or their monomers produce electrical signals for their recognition;

[0075] 4) A system for direct electronic identification and / or sequencing of biopolymers, comprising:

[0076] a. One of the devices described in 1) to 3);

[0077] b. Mechanisms for controlling the movement of biopolymers through nanopores or nanochannels with sub-nanometer precision;

[0078] c. a bias source for applying a bias between the cis chamber and the trans chamber to guide one end of the biopolymer into the nanopore;

[0079] d. a voltage source for applying a voltage between the two electrodes to measure the current fluctuation through the bridged molecule;

[0080] e. Software for signal acquisition and analysis.

[0081] The biopolymer may be DNA, RNA, protein, peptide, polysaccharide, etc.; the motion control mechanism may be piezoelectric drive, electromechanical drive, magnetic or electric mode, or any combination thereof, such as the control mechanism capable of achieving sub-nanometer precision described in PCT WO2017075620A1; the above system may have a scanning plate, the biopolymer may be attached to the scanning plate by chemical binding or by magnetic beads, and the scanning plate is moved by a nano-precision actuator. The controlled movement speed of each monomer or base is 0.1ms to 1000ms, preferably 1ms to 100ms, and most preferably 5ms to 20ms.

[0082] 5) Methods for identification and / or sequencing of biopolymers

[0083] a. Providing one of the devices described in 1) to 2);

[0084] b. Functionalizing electrodes to facilitate attachment of molecular modules; using different electrode materials and attachment chemistries to attach molecular modules in a controlled manner;

[0085] c. Connecting the first end of the biopolymer to the magnetic bead directly or through a linker molecule using the method described in PCT patent WO2017075620A1;

[0086] d. Aligning the biopolymer with the nanopore equipped with a bridging molecule and the molecular probe using one of the methods described in PCT patent WO20175620A1;

[0087] e. directing the second end of the biopolymer to the nanopore by electrophoretic force;

[0088] f. Moving biopolymers through nanopores in a precisely controlled manner as described in PCT patent WO20175620A1,

[0089] g. Apply a bias voltage to the two electrodes and measure the current fluctuation caused by the interaction between the molecular module and the biopolymer.

[0090] 6) Methods for identification and / or sequencing of biopolymers:

[0091] a. Providing the device described in 3);

[0092] b. Functionalizing electrodes to facilitate attachment of molecular modules; using different electrode materials and attachment chemistries to attach molecular modules in a controlled manner;

[0093] c. attaching the first end of the biopolymer to the bead or nanoparticle directly or through a molecular linker;

[0094] d. passing biopolymers through nanochannels by fluid flow or electrophoretic forces;

[0095] e. Apply a bias voltage to the two electrodes and measure the current fluctuations as the biopolymer passes through the nanogap.

[0096] The molecular module consists of a conductive part and a capture (cleft or tweezers) part;

[0097] wherein the capture moiety is

[0098] a) Planar structures that can interact with biopolymers through hydrogen bonding or aromatic stacking

[0099] b) A moiety consisting of two planar structures linked together by a spacer molecule, which can interact with the chemical entity through hydrogen bonding, aromatic stacking, or hydrogen bonding and aromatic stacking.

[0100] c) Structures that interact with DNA or RNA nucleobases through hydrogen bonding or base stacking.

[0101] d) A molecular cleft that traps DNA or RNA nucleobases in its hollow space.

[0102] e) Molecular tweezers or clamps that capture DNA or RNA nucleobases through hydrogen bonding and base stacking.

[0103] f) Those that can distinguish between nucleobases by forming different non-covalent complexes with different binding energies.

[0104] The conductive part includes

[0105] a) Organic molecules whose conformation can be changed and thus their conductivity. In particular, these molecules contain aromatic structures.

[0106] b) Natural nucleic acids that have a double helix structure and have GC base pairs.

[0107] c) Natural nucleic acids having a double helix structure and containing modified bases, modified nucleosides or modified nucleotides

[0108] d) DNA analogs that form a double helix structure and contain natural nucleobases.

[0109] e) DNA analogs that form double helical structures and contain non-natural nucleobases.

[0110] f) DNA analogs that contain unnatural bases and form a double helix structure.

[0111] g) DNA analogs composed of non-phosphate backbones

[0112] h) Peptides having an α-helix or β-sheet structure and containing aromatic amino acids and charged amino acids.

[0113] i) Peptides comprising non-natural aromatic amino acids.

[0114] j) Peptides composed of non-amide backbones

[0115] The one or more molecular wires in the above device have a length of 2 nm to 1 μm, and the ends thereof contain anchor molecules that can react with electrodes to form covalent bonds.

[0116] 1), 2) and 3) in the device described in the electrode, which is

[0117] a) A metal electrode that can directly react with the anchoring molecule.

[0118] b) Metal electrodes that may be functionalized on their surface by a self-assembled monolayer that can react with anchoring molecules to form covalent bonds.

[0119] c) Metal oxide electrodes can be functionalized with silanes, which can react with anchor molecules to form covalent bonds.

[0120] d) Carbon electrodes can be functionalized with organic reagents, which can react with anchor molecules to form covalent bonds.

[0121] The anchoring molecule described herein is

[0122] a) Those containing one or more amino groups.

[0123] b) Those comprising one or more thiol groups.

[0124] c) Those containing one or more disulfide groups.

[0125] d) Those comprising one or more diol groups.

[0126] The monolayer is formed by

[0127] a) Identical organic molecules, each with a thiol group 0.2 to 3 nm long.

[0128] b) Identical organic molecules, each with multiple thiol groups 0.2 to 3 nm long.

[0129] c) The same organic molecule as described in 9-a and 9-b, which contains a functional group that can react with the anchoring group in claim 8

[0130] d) Thiol tripods, which form a monolayer on the metal surface and react with the anchoring molecules.

[0131] e) A mixture of the organic reagent or a mixture of the organic molecule and a thiol tripod.

[0132] General Notes:

[0133] All publications, patent applications, patents and other documents mentioned herein are incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the invention belongs. Although the invention is introduced by embodiments of the invention, and these embodiments are described in considerable detail, they should not be construed to limit or in any way limit the scope of the appended claims. Other advantages and modifications will be readily apparent to those skilled in the art. Therefore, the invention is not limited to the specific details, representative devices, devices and methods and illustrative examples shown and described in its broader aspects. Therefore, departing from these details does not necessarily depart from the spirit of the invention.

[0134] References

[0135] 1. Armentano, D.; De Munno, G.; Di Donna, L.; Sindona, G.; Giorgi, G.; Salvini,

[0136] L.; Napoli, A., Cytosine self-assembles into triply bound dimers in acidic medium

[0137] (Self-assembling of cytosine nucleoside into triply-bound dimers inacid media). A comprehensive evaluation of proton-bound pyrimidine nucleosides by electrospray tandem mass spectrometry, X-rays diffractometry, and theoretical calculations. Journal of the American Society for Mass Spectrometry 2004, 15, 268-79.

[0138] 2. Goodwin, S.; McPherson, JD; McCombie, WR. Coming of age: ten years of next-generation sequencing technologies. Nature reviews. Genetics 2016, 17, 333-51.

[0139] 3. Metzker, ML, Emerging technologies in DNA sequencing. Genome Res 2005, 15, 1767-76.

[0140] 4. Treangen, TJ; Salzberg, SL. Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nature reviews. Genetics 2012, 13, 36-46.

[0141] 5. Rhoads, A.; Au, KF, PacBio Sequencing and Its Applications. Genomics Proteomics Bioinformatics 2015, 13, 278-89.

[0142] 6. Gordon, D.; Huddleston, J.; Chaisson, M. P.; Hill, C. M.; Kronenberg, Z. N.; Munson, K. M.; Malig, M.; Raja, A.; Fiddes, I.; Hillier, L. W.; Dunn, C.; Baker, C.; Armstrong, J.; Diekhans, M.; Paten, B.; Shendure, J.; Wilson, R. K.; Haussler, D.; Chin, C.-S.; Eichler, E. E. Long-read sequence assembly of the gorilla genome. Science 2016, 352, aae0344-1.

[0143] 7. Mukhopadhyay, R., DNA sequencers: the next generation. Analytical Chemistry 2009, 8, 1736-1740.

[0144] 8. Deamer, D.; Akeson, M.; Branton, D., Three decades of nanopore sequencing. Nat Biotechnol 2016, 34, 518-24.

[0145] 9. Jain, M.; Fiddes, I.T.; Miga, K.H.; Olsen, HE.; Paten, B.; Akeson, M., Improved data analysis for the MinION nanoporesequencer. Nature methods 2015, 12, 351-6.

[0146] 10. Manrao, EA; Derrington, IM; Laszlo, AH; Langford, KW; Hopper, MK; Gillgren, N.; Pavlenok, M.; Niederweis, M.; Gundlach, JH. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotechnol 2012, 30, 349-353.

[0147] 11. Laszlo, AH; Derrington, IM; Ross, BC; Brinkerhoff, H.; Adey, A.; Nova, IC; Craig, JM; Langford, KW; Samson, JM; Daza, R.; Doering, K.; Shendure, J.; Gundlach, JH. Decoding long nanopore sequencing reads of natural DNA. Nature Biotechnology 2014, 32, 829-833.

[0148] 12. Lindsay, S., The promises and challenges of solid-state sequencing. Nat Nanotechnol 2016, 11, 109-11.

[0149] 13. Lagerqvist, J.; Zwolak, M.; Ventra, MD, Fast DNA Sequencing via Transverse Electronic Transport. Nano Lett 2006, 6, 779-782.

[0150] 14. Di Ventra, M.; Taniguchi, M., Decoding DNA, RNA and peptides with quantum tunnelling. Nat Nanotechnol 2016, 11, 117-26.

[0151] 15. Tsutsui, M.; Taniguchi, M.; Yokota, K.; Kawai, T., Identifying single nucleotides by tunnelling current. Nat Nanotechnol 2010, 5, 286-90.

[0152] 16. Tsutsui, M.; Rahong, S.; Iizumi, Y.; Okazaki, T.; Taniguchi, M.; Kawai, T., Single-molecule sensing electrode embedded in-plane nanopore. Scientific reports 2011, 1, 46.

[0153] 17. Alvarez, JR; Skachkov, D.; Massey, SE; Kalitsov, A.; Velev, JP,

[0154] DNA / RNA transverse current sequencing: intrinsic structural noise from neighboring bases. Frontiers in genetics 2015, 6, 213.

[0155] 18. Prasongkit, J.; Feliciano, GT; Rocha, AR; He, Y.; Osotchan, T.; Ahuja, R.; Scheicher, RH. Theoretical assessment of feasibility to sequence DNA through interlayer electronic tunneling transport at aligned nanopores inbilayer graphene. Scientific reports 2015, 5, 17560.

[0156] 19. Biswas, S.; Sen, S.; Im, J.; Biswas, S.; Krstic, P.; Ashcroft, B.; Borges, C.; Zhao, Y.; Lindsay, S.; Zhang, P., Universal Readers Based on Hydrogen Bonding or pi-pi Stacking for Identification of DNA Nucleotides in Electron Tunnel Junctions. ACS Nano 2016, 10, 11304-11316.

[0157] 20. Olsen, TJ; Choi, Y.; Sims, P.C.; Gul, O.T.; Corso, B.L.; Dong, C.; Brown, W.A.; Collins, P.G.; Weiss, G.A. Electronic measurements of single-molecule processing by DNA polymerase I (Klenow fragment). J Am Chem Soc 2013, 135, 7855-60.

[0158] 21. Pugliese, KM; Gul, O.T; Choi, Y.; Olsen, T.J.; Sims, P.C.; Collins, P.G.; Weiss, G.A. Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits. J Am Chem Soc 2015, 137, 9587-94.

[0159] 22. Xie, P.; Xiong, Q.; Fang, Y.; Qing, Q.; Lieber, CM. Local electrical potential detection of DNA by nanowire–nanopore sensors. Nature Nanotechnology 2012, 7, 119-125.

[0160] 23. Nakatsuka, N.; Yang, K.-A.; Abendroth, JM; Cheung, KM; Xu, X.; Yang, H.; Zhao, C.; Zhu, B.; Rim, YS; Yang, Y.; Weiss, PS; MN; Andrews, AM, Aptamer–field-effect transistors overcome Debye length limitations for small-molecule sensing. Science 2018, 362, 319–324.

[0161] 24. Wang, G.; Kim, T.-W.; Jang, Y. H.; Lee, T., Effects of Metal-Molecule Contact and Molecular Structure on Molecular Electronic Conduction in Nonresonant Tunneling Regime: Alkyl versus Conjugated Molecules. J. Phys. Chem. C 2008, 112, 13010–13016.

[0162] 25. Xin, N.; Wang, J.; Jia, C.; Liu, Z.; Zhang, X.; Yu, C.; Li, M.; Wang, S.; Gong, Y.; Sun, H.; Zhang, G.; Liu, Z.; Zhang, G.; Liao, J.; Zhang, D.; Guo, X., Stereoelectronic Effect-Induced Conductance Switching in Aromatic Chain Single-Molecule Junctions. Nano Lett. 2017, 17, 856-861.

[0163] 26. Fujii, S.; Tada, T.; Komoto, Y.; Osuga, T.; Murase, T.; Fujita, M.; Kiguchi, M., Rectifying Electron-Transport Properties through Stacks of Aromatic Molecules Inserted into a Self-Assembled Cage. J Am Chem Soc 2015, 137, 5939-47.

[0164] 27. Chen, Y.-S.; Hong, M.-Y.; Huang, G.S., A protein transistor made of an antibody molecule and two gold nanoparticles. Nature Nanotechnology 2012, 7, 197-203.

[0165] 28.Amdursky, N., Electron Transfer across Helical Peptides. ChemPlusChem 2015, 80, 1075-1095.

[0166] 29. Reguera, G.; McCarthy, KD; Mehta, T.; Nicoll, JS; Tuominen, MT; Lovley, DR. Extracellular electron transfer via microbial nanowires. Nature 2005, 435, 1098-101.

[0167] 30. Petrov, EG; Shevchenko, YV; Teslenko, VI; May, V., Nonadiabatic donor–acceptor electron transfer mediated by a molecular bridge: A unified theoretical description of the superexchange and hopping mechanism. The Journal of chemical physics 2001, 115, 7107-7122.

[0168] 31. Malak, RA; Gao, Z.; Wishart, JF; Isied, SS. Long-Range Electron Transfer Across Peptide Bridges: The Transition from Electron Superexchange to Hopping. J. AM. CHEM. SOC. 2004, 126, 13888-13889.

[0169] 32. Qing, Q.; Wang, Y.; Sadar, J. NANOPOREDEVICES FOR SENSING BIOMOLECULES. 2018, US2018 / 0280968.

[0170] 33. Sadara, J.; Wang, Y.; Qing, Q., Confined Electrochemical Deposition in Sub-15nm Space for Preparing Nanogap Electrodes. ECS Transactions 2017, 77, 65-72

[0171] 34. Ing, NL; Spencer, RK; Luong, SH; Nguyen, HD; Hochbaum, AI. Electronic Conductivity in Biomimetic alpha-Helical Peptide Nanofibers and Gels. ACS Nano 2018, 12, 2652-2661.

[0172] 35. Raliski, BK; Howard, CA; Young, DD. Site-Specific Protein Immobilization Using Unnatural Amino Acids. Bioconjug Chem 2014, 25, 1916–1920.

[0173] 36. Kalyoncu, E.; Ahan, R. E.; Olmez, TT; Safak Seker, U. O. Genetically encoded conductive protein nanofibers secreted by engineered cells. RSC Advances 2017, 7, 32543-32551.

[0174] 37. Morera, E.; Pinnen, F.; Lucente, G., Synthesis of 1,2-Dithiolane Analogues of Leucine for Potential Use in Peptide Chemistry. Organic Letters 2002, 4, 1139-1142.

[0175] 38. Staderini, M.; González-Fernández, E.; Murray, AF; Mount, AR; Bradley, M., A tripod anchor offers improved robustness of peptide-based electrochemical biosensors. Sensors and Actuators B: Chemical 2018, 274, 662-667.

[0176] 39. Lee, SU; Mizuseki, H.; Kawazoe, Y., Rigid adamantane tripod linkage for well-defined conductance of a single-molecule junction. Physical chemistry chemical physics: PCCP 2010, 12, 11763-9.

[0177] 40. Meadows, MK; Roesner, EK; Lynch, VM; James, TD; Anslyn, EV. Boronic Acid Mediated Coupling of Catechols and N-Hydroxylamines: A Bioorthogonal Reaction to Label Peptides. Org Lett 2017, 19, 3179-3182.

[0178] 41. Neubert, T. J.; F.; Sun, G.; Janietz, S.; Gluba, M.A.; Hinrichs, K.; Nickel, N.H.; Rappich, J., Functionalization of gold and graphene electrodes by p-maleimido-phenyl towards thiol-sensing systems investigated by EQCM and IR ellipsometric spectroscopy. Applied Surface Science 2017, 421, 755-760.

[0179] 42. Tong, L.; Gothelid, M.; Sun, L., Oxygen evolution at functionalized carbon surfaces: a strategy for immobilization of molecular water oxidation catalysts. Chem Commun 2012, 48, 10025-7.

[0180] 43. Kerwin, S.; Reinus, B., A Copper-Catalyzed N-Alkynylation Route to 2-Substituted N-Alkynyl Pyrroles and Their Cyclizationinto Pyrrolo[2,1-c]oxazin-1-ones: A Formal Total Synthesis of Peramine. Synthesis 2017, 49, 2544-2554.

[0181] 44. Kitagawa, T.; Idomoto, Y.; Matsubara, H.; Hobara, D.; Kakiuchi, T.; Okazaki, T.; Komatsu, K., Rigid Molecular Tripod with an Adamantane Frameworkand Thiol Legs. Synthesis and Observation of an Ordered Monolayer on Au(111). J. Org. Chem. 2006, 71, 1362-1369.

[0182] 45.Ohsawa, K.; Yoshida, M.; Doi, T., A direct and mild formylation method for substituted benzenes utilizing dichloromethyl methyl ether-silvertrifluoromethanesulfonate. J Org Chem 2013, 78, 3438-44.

[0183] 46. ​​Mfuh, A. M.; Doyle, J. D.; Chhetri, B.; Arman, H. D.; Larionov, O. V. Scalable, Metal- and Additive-Free, Photoinduced Borylation of Haloarenes and Quaternary Arylammonium Salts. J Am Chem Soc 2016, 138, 2985-2988.

Claims

1. A system for electronically identifying and / or sequencing a biopolymer, comprising: a. A substrate placed between the cis reservoir and the trans reservoir; b. nanopores in the substrate that allow at least a portion of the biopolymer to be transferred from the cis reservoir to the trans reservoir; c. a first electrode and a second electrode embedded in the nanopore and overlapping each other, which are separated by a dielectric layer to form a nanogap therebetween; d. A synthetic T-shaped or Y-shaped molecular module having a conductive molecular wire with an anchoring molecule at its end for connection, which can bridge two electrodes and allow current to flow from one electrode to another electrode; and a receptor that can interact with a biopolymer or its constituent monomers for sensing, wherein when the molecular module is a T-shaped molecular module, the receptor is a molecular cleft comprising two aromatic motifs coupled to a pyridine molecule, and wherein when the molecular module is a Y-shaped molecular module, the receptor is a molecular tweezer comprising two different aromatic motifs coupled to a five-membered aromatic ring; e. a mechanism configured to control the speed at which the biopolymer moves through the nanopore; f. a first bias source for applying a bias between the cis reservoir and the trans reservoir to promote the movement of the biopolymer through the nanopore; g. a second bias source for applying a bias voltage between the two electrodes; and h. A measurement system configured to measure and analyze a signal when a biopolymer passes through a nanopore that affects an electric current, and to identify the biopolymer or its constituent monomers based on the signal.

2. A system for electronically identifying and / or sequencing a biopolymer, comprising: a. A substrate placed between the cis reservoir and the trans reservoir; b. nanopores in the substrate that allow at least a portion of the biopolymer to be transferred from the cis reservoir to the trans reservoir; c. A pair of electrodes separated by a nanogap on the same plane of the nanopore surface, either on the inlet side or on the outlet side, wherein the nanogap is located at the center of the nanopore, off-center, or along the edge of the nanopore; d. A synthetic T-shaped or Y-shaped molecular module having a conductive molecular wire with an anchoring molecule at its end for connection, which can bridge two electrodes and allow current to flow from one electrode to another electrode; and a receptor that can interact with a biopolymer or its constituent monomers for sensing, wherein when the molecular module is a T-shaped molecular module, the receptor is a molecular cleft comprising two aromatic motifs coupled to a pyridine molecule, and wherein when the molecular module is a Y-shaped molecular module, the receptor is a molecular tweezer comprising two different aromatic motifs coupled to a five-membered aromatic ring; e. a mechanism configured to control the speed at which the biopolymer moves through the nanopore; f. a first bias source for applying a bias between the cis reservoir and the trans reservoir to promote the movement of the biopolymer through the nanopore; g. a second bias source for applying a bias voltage between the two electrodes; and h. A measurement system configured to measure and analyze a signal when a biopolymer passes through a nanopore that affects an electric current, and to identify the biopolymer or its constituent monomers based on the signal.

3. A system for electronically identifying and / or sequencing a biopolymer, comprising: a. A planar substrate having a cis reservoir and a trans reservoir; b. a nanochannel in a substrate that allows at least a portion of a biopolymer to move from a cis reservoir to a trans reservoir through the nanochannel, wherein the nanochannel gradually decreases its size continuously or stepwise and has or does not have microstructures on the bottom or walls of the nanochannel, which are configured to stretch and slow down the progress of the biopolymer; c. A first electrode and a second electrode are positioned opposite to each other at the end of the nanochannel, the first electrode is embedded in the wall of one side of the nanochannel, and the second electrode is embedded in the wall of the other side of the nanochannel, forming a nanogap therebetween, and the gap size is equal to the local width of the nanochannel; or, both electrodes are embedded in the wall of the same side of the nanochannel, with a dielectric layer therebetween, forming a nanogap therebetween, and the gap size is equal to the thickness of the dielectric layer; d. A synthetic T-shaped or Y-shaped molecular module having a conductive molecular wire with an anchoring molecule at its end for connection, which can bridge two electrodes and allow current to flow from one electrode to another electrode; and a receptor that can interact with a biopolymer or its constituent monomers for sensing, wherein when the molecular module is a T-shaped molecular module, the receptor is a molecular cleft comprising two aromatic motifs coupled to a pyridine molecule, and wherein when the molecular module is a Y-shaped molecular module, the receptor is a molecular tweezer comprising two different aromatic motifs coupled to a five-membered aromatic ring; e. a mechanism configured to control the speed at which the biopolymer moves through the nanochannel; f. a first bias source for applying a bias between the cis reservoir and the trans reservoir to promote the movement of the biopolymer through the nanochannel; g. a second bias source for applying a bias voltage between the two electrodes; and h. A measurement system configured to measure and analyze a signal when a biopolymer passes through a nanochannel that affects an electric current, and to identify the biopolymer or its constituent monomers based on the signal.

4. A system as claimed in claim 1, 2 or 3, wherein Biopolymers are DNA, RNA, proteins, peptides, polysaccharides or combinations thereof, whether natural, modified or synthetic.

5. The system of claim 4, wherein The biopolymers are oligonucleotides.

6. The system of claim 1, 2 or 3, wherein The substrate is a nanochip containing a plurality of nanoholes or nanochannels.

7. The system of claim 1, 2 or 3, wherein Mechanisms configured to control the speed at which a biopolymer moves through a nanopore or nanochannel include: a. A scanning plate placed parallel to the substrate, to which the biopolymer is directly or indirectly attached; and b. An actuator for controlling the relative movement between the substrate and the scanning plate with nanometer precision; wherein the relative movement is in the normal direction or in the tangential direction, wherein the relative movement in the normal direction refers to changing the distance between the scanning plate and the substrate, and the relative movement in the tangential direction refers to lateral movement without changing the distance between the scanning plate and the substrate.

8. The system of claim 7, wherein: Actuators are precision linear motion stages driven by the piezoelectric effect, electromechanical actuation, or any method that uses magnetic, electrical, or mechanical elements, or a combination of these.

9. The system of claim 7, wherein: The biopolymer is directly linked to the scanning plate through chemical bonds, or indirectly linked to the scanning plate through magnetic beads or linker molecules, or a combination thereof.

10. The system of claim 9, wherein: The chemical bond is a covalent bond or a non-covalent bond.

11. A system as claimed in claim 9 or 10, wherein: The linker molecule is selected from single-stranded nucleic acid, double-stranded nucleic acid, polypeptide chain, cellulose fiber or any flexible linear polymer and combinations thereof, and can be natural, modified or synthetic.

12. The system of claim 1, 2 or 3, wherein Mechanisms configured to control the speed at which a biopolymer moves through a nanopore or nanochannel include enzymes, any medium or microstructure that can slow the speed at which a biopolymer moves through it, or electrophoretic forces configured to slow the movement of a biopolymer, or a combination thereof.

13. The system of claim 12, wherein The enzyme is an enzyme with a conditioned medium that can slow down the activity of the enzyme.

14. The system of claim 12, wherein Any medium that can slow down the rate at which a biopolymer moves through it is either a viscous medium or a porous medium.

15. The system of claim 12, wherein: The enzyme is a DNA or RNA polymerase or a helicase.

16. The system according to any one of claims 1 to 3, wherein The controlled migration speed of the biopolymers ranged from 0.1 ms to 1000 ms per monomer.

17. The system of claim 16, wherein The controlled migration speed of the biopolymers ranged from 1 ms to 100 ms per monomer.

18. The system of claim 16, wherein The controlled migration speed of the biopolymers ranged from 5 ms to 20 ms per monomer.

19. The system of claim 1, 2 or 3, wherein The molecular wire includes one or a combination of the following: a) Molecules whose conductivity changes with conformation; b) natural nucleic acids having a double helical structure and comprising mainly GC base pairs; c) a natural nucleic acid having a double helix structure, which comprises a modified base, a modified nucleoside or a modified nucleotide; d) DNA that forms a double helix structure, which contains natural nucleobases; e) DNA that forms a double helix structure, which contains non-natural or synthetic nucleobases; f) DNA composed of non-natural or synthetic bases and forming a double helix structure; g) DNA comprising a non-phosphate backbone; h) peptides comprising an α-helix or β-sheet structure and containing aromatic amino acids and charged amino acids; i) a peptide comprising non-natural or synthetic aromatic amino acids; or j) peptides comprising a non-amide backbone; And the receptors include one or a combination of the following: a) a planar structure that can interact with biopolymers through hydrogen bonding or aromatic stacking; b) a moiety consisting of two planar structures linked together by a spacer molecule, which can interact with the chemical entity through hydrogen bonding, aromatic stacking, or hydrogen bonding and aromatic stacking; c) structures that interact with DNA or RNA nucleobases through hydrogen bonding or base stacking; d) a molecular cleft that traps DNA or RNA nucleobases in its hollow space; e) molecular tweezers or clamps that capture DNA or RNA nucleobases through hydrogen bonding and base stacking; or f) Any molecule that can distinguish between nucleobases by forming different non-covalent complexes with different binding energies.

20. The system of claim 1, 2 or 3, wherein The anchoring molecules include the following or a combination thereof: a) molecules containing one or more amino groups; b) molecules comprising one or more thiol groups; c) a molecule comprising one or more disulfide groups; or d) Molecules comprising one or more diol groups.

21. The system of claim 1, 2 or 3, wherein Anchor molecules contain three prongs, or legs, which form a stable and strong connection of the molecular wire to the electrode.

22. The system of claim 1, 2 or 3, wherein The two electrodes have the same or different materials, including the following or a combination thereof: a) a metal electrode that can directly react with the anchoring molecule; b) can react with anchor molecules to form a covalently bonded self-assembled monolayer A metal electrode functionalized on its surface; c) a metal oxide electrode that can be functionalized with silanes, which can react with the anchoring molecule to form a covalent bond; or d) Carbon electrodes can be functionalized with organic reagents, which can react with anchor molecules to form covalent bonds.

23. The system of claim 22, wherein: The single layer is formed by one or a combination of the following: a) an organic molecule containing a thiol group, 0.2 to 3 nm long; b) an organic molecule comprising a plurality of thiol groups, 0.2 to 3 nm in length; c) an organic component comprising a functional group that can react with the metal-specific anchoring molecule son; d) thiol tripods, which form a monolayer on the metal surface and react with metal-specific anchoring molecules; or e) A mixture of the organic molecules or a mixture of the organic molecules and a thiol tripod.

24. The system of claim 1, 2 or 3, further comprising One or more additional pairs of electrodes embedded in the nanopore or nanochannel are used to measure the biopolymer multiple times as the biopolymer passes through the nanopore or nanochannel.

25. The system of claim 1, 2 or 3, wherein The size of the nanogap separating the two electrodes is 3nm to 1um, and the length of the molecular wire is comparable to the nanogap, facilitating the formation of a bridge between the electrode pair.

26. The system of claim 25, wherein The size of the nanogap separating the two electrodes is 5nm to 20nm.

27. A method for electronically identifying and / or sequencing a biopolymer, comprising: a. Providing a substrate between the cis reservoir and the trans reservoir; b. providing a nanopore in the substrate that allows at least a portion of the biopolymer to be transferred from the cis reservoir to the trans reservoir; c. providing a first electrode and a second electrode embedded in the nanopore and overlapping each other, which are separated by a dielectric layer to form a nanogap therebetween; d. providing a synthetic T-shaped or Y-shaped molecular module having a conductive molecular wire with an anchor molecule at its end for connection, which can bridge two electrodes; and a receptor that can interact with a biopolymer or its constituent monomers for sensing, wherein when the molecular module is a T-shaped molecular module, the receptor is a molecular cleft comprising two aromatic motifs coupled to a pyridine molecule, and wherein when the molecular module is a Y-shaped molecular module, the receptor is a molecular tweezer comprising two different aromatic motifs coupled to a five-membered aromatic ring; e. Connecting the molecular module to two electrodes, with one end connected to the first electrode and the other end connected to the second electrode, so that current flows from one electrode to the other electrode; f. providing a mechanism configured to control the speed at which the biopolymer moves through the nanopore; g. providing a first bias source for applying a bias between the cis reservoir and the trans reservoir to promote entry and translocation of the biopolymer through the nanopore; h. providing a second bias source for applying a bias voltage between the two electrodes; and i. Provide a measurement system that can measure and analyze the signal when a biopolymer passes through a nanopore that affects an electric current, and identify the biopolymer or its constituent monomers based on the signal.

28. A method for electronically identifying and / or sequencing a biopolymer, comprising: a. Providing a substrate disposed between the cis reservoir and the trans reservoir; b. providing a nanopore in the substrate that allows at least a portion of the biopolymer to be transferred from the cis reservoir to the trans reservoir; c. Providing a pair of electrodes separated by a nanogap on the same plane of the nanopore surface, either on the inlet side or on the outlet side, wherein the nanogap is located at the center of the nanopore, off-center, or along the edge of the nanopore; d. providing a synthetic T-shaped or Y-shaped molecular module having a conductive molecular wire with an anchor molecule at its end for connection, which can bridge two electrodes; and a receptor that can interact with a biopolymer or its constituent monomers for sensing, wherein when the molecular module is a T-shaped molecular module, the receptor is a molecular cleft comprising two aromatic motifs coupled to a pyridine molecule, and wherein when the molecular module is a Y-shaped molecular module, the receptor is a molecular tweezer comprising two different aromatic motifs coupled to a five-membered aromatic ring; e. Connecting the molecular module to two electrodes, with one end connected to the first electrode and the other end connected to the second electrode, so that current flows from one electrode to the other electrode; f. provide a mechanism to control the speed at which biopolymers move through nanopores; g. providing a first bias source for applying a bias between the cis reservoir and the trans reservoir to promote entry and translocation of the biopolymer through the nanopore; h. providing a second bias source for applying a bias voltage between the two electrodes; and i. Provide a measurement system that can measure and analyze the signal when a biopolymer passes through a nanopore that affects an electric current, and identify the biopolymer or its constituent monomers based on the signal.

29. A method for electronically identifying and / or sequencing a biopolymer, comprising: a. Providing a planar substrate having a cis reservoir and a trans reservoir; b. providing a nanochannel in a substrate that allows at least a portion of a biopolymer to move from a cis reservoir to a trans reservoir through the nanochannel, wherein the nanochannel gradually decreases its size continuously or stepwise, and has or does not have microstructures on the bottom or walls of the nanochannel that help stretch and slow down the biopolymer; c. providing a first electrode and a second electrode, which are positioned opposite to each other at the end of the nanochannel, the first electrode being embedded in the wall on one side of the nanochannel, and the second electrode being embedded in the wall on the other side of the nanochannel, forming a nanogap therebetween, and the gap size being equal to the local width of the nanochannel; or, both electrodes being embedded in the wall on the same side of the nanochannel, with a dielectric layer therebetween, forming a nanogap therebetween, and the gap size being equal to the thickness of the dielectric layer; d. providing a synthetic T-shaped or Y-shaped molecular module having a conductive molecular wire with an anchoring molecule at its end for connection, which can bridge two electrodes; and a receptor that can interact with a biopolymer or its constituent monomers for sensing, wherein when the molecular module is a T-shaped molecular module, the receptor is a molecular cleft comprising two aromatic motifs coupled to a pyridine molecule, and wherein when the molecular module is a Y-shaped molecular module, the receptor is a molecular tweezer comprising two different aromatic motifs conjugated to a five-membered aromatic ring; e. Connecting the molecular module to two electrodes, with one end connected to the first electrode and the other end connected to the second electrode, so that current flows from one electrode to the other electrode; f. provide a mechanism to control the speed at which biopolymers move through nanochannels; g. providing a first bias source for applying a bias between the cis reservoir and the trans reservoir to promote entry and movement of the biopolymer through the nanochannel; h. providing a second bias source for applying a bias voltage between the two electrodes; and i. Provide a measurement system that can measure and analyze the signal when a biopolymer passes through a nanochannel that affects an electric current, and identify the biopolymer or its constituent monomers based on the signal.

30. The method of claim 27, 28 or 29, wherein Biopolymers are DNA, RNA, proteins, peptides, polysaccharides or combinations thereof, whether natural, modified or synthetic.

31. The method of claim 30, wherein The biopolymers are oligonucleotides.

32. The method of claim 27, 28 or 29, wherein The substrate is a nanochip containing a plurality of nanoholes or nanochannels.

33. The method of claim 27, 28 or 29, wherein Providing a mechanism for controlling the speed at which a biopolymer moves through a nanopore or nanochannel, further comprising: a. providing a scanning plate placed parallel to the substrate, the biopolymer is directly or indirectly attached thereto; b. Providing an actuator for controlling the relative movement between the substrate and the scanning plate with nanometer precision; wherein the relative movement is in the normal direction or in the tangential direction, wherein the relative movement in the normal direction refers to changing the distance between the scanning plate and the substrate, and the relative movement in the tangential direction refers to lateral movement without changing the distance between the scanning plate and the substrate; c. directly connecting the biopolymer to the scanning plate through a chemical bond, or indirectly connecting to the scanning plate through a magnetic bead or a linker molecule, or a combination thereof; d. directing one end of the biopolymer into the nanopore or nanochannel using a first bias source; and e. Using nanometer-precision actuators to change the relative position between the scanning plate and the substrate, the biopolymer moves through the nanopore or nanochannel at a slow and controllable speed.

34. The method of claim 33, wherein the chemical bond is a covalent bond or a non-covalent bond.

35. The method of claim 33, wherein Actuators are precision linear motion stages driven by piezoelectric effect, electromechanical drive, or any combination of magnetic, electrical or mechanical elements.

36. The method of claim 33, wherein The linker molecule is selected from single-stranded nucleic acid, double-stranded nucleic acid, polypeptide chain, cellulose fiber or any flexible linear polymer and combinations thereof, and can be natural, modified or synthetic.

37. The method of claim 27, 28 or 29, wherein The controlled migration speed of biopolymers ranges from 0.1 ms to 1000ms.

38. The method of claim 37, wherein The controlled migration speed of biopolymers is 1 ms to 100 ms per monomer or base.

39. The method of claim 37, wherein The controlled migration speed of biopolymers is 5 ms to 20 ms per monomer or base.

40. The method of claim 27, 28 or 29, wherein The molecular wire is composed of one or a combination of the following: a) Molecules whose conductivity changes with conformation; b) natural nucleic acids having a double helical structure and comprising mainly GC base pairs; f) a natural nucleic acid having a double helix structure, which comprises a modified base, a modified nucleoside or a modified nucleotide; g) DNA that forms a double helix structure, which comprises natural nucleobases; h) a DNA that forms a double helix structure, comprising a non-natural nucleobase; i) DNA composed of unnatural bases and forming a double helix structure; j) DNA composed of a non-phosphate backbone; k) peptides having an α-helix or β-sheet structure and containing aromatic amino acids and charged amino acids; 1) peptides comprising non-natural aromatic amino acids; m) peptides consisting of a non-amide backbone; And the receptors include one or a combination of the following: a) a planar structure that can interact with biopolymers through hydrogen bonding or aromatic stacking; b) a moiety consisting of two planar structures linked together by a spacer molecule, which can interact with the chemical entity through hydrogen bonding, aromatic stacking, or hydrogen bonding and aromatic stacking; c) structures that interact with DNA or RNA nucleobases through hydrogen bonding or base stacking; d) a molecular cleft that traps DNA or RNA nucleobases in its hollow space; e) Molecular tweezers or clamps that capture DNA or RNA nucleobases through hydrogen bonding and base stacking; f) Any molecule that can distinguish between nucleobases by forming different non-covalent complexes with different binding energies.

41. The method of claim 27, 28 or 29, wherein Anchor molecules with three prongs or legs that form a more stable and secure connection of the molecular wire to the electrode.

42. The method of claim 27, 28 or 29, wherein The anchoring molecules include the following or a combination thereof: a) molecules containing one or more amino groups; b) molecules comprising one or more thiol groups; c) molecules containing one or more disulfide groups; d) Molecules comprising one or more diol groups.

43. The method of claim 27, 28 or 29, wherein The two electrodes have the same or different materials, including the following or a combination thereof: a) a metal electrode that can directly react with the anchoring molecule; b) can react with anchor molecules to form a covalently bonded self-assembled monolayer A metal electrode functionalized on its surface; c) a metal oxide electrode that can be functionalized with silanes, which can react with the anchoring molecule to form a covalent bond; or d) Carbon electrodes can be functionalized with organic reagents, which can react with anchor molecules to form covalent bonds.

44. The method of claim 43, wherein The single layer is formed by one or a combination of the following: a) an organic molecule containing a thiol group, 0.2 to 3 nm long; b) an organic molecule comprising a plurality of thiol groups, 0.2 to 3 nm in length; c) an organic component comprising a functional group that can react with the metal-specific anchoring molecule son; d) thiol tripods, which form a monolayer on the metal surface and react with metal-specific anchoring molecules; or e) A mixture of the organic molecules or a mixture of the organic molecules and a thiol tripod.

45. The method of claim 27, 28 or 29, wherein The size of the nanogap separating the two electrodes is 3nm to 1um, and the length of the molecular wire is comparable to the nanogap, facilitating the formation of a bridge between the electrode pair.

46. ​​The method of claim 45, wherein The size of the nanogap separating the two electrodes is 5nm to 20nm.

47. The system of claim 13 or 14, wherein: The enzyme is a DNA or RNA polymerase or a helicase.

48. The method of claim 34, wherein Actuators are precision linear motion stages driven by piezoelectric effect, electromechanical drive, or any combination of magnetic, electrical or mechanical elements.

49. The method of claim 34, wherein The linker molecule is selected from single-stranded nucleic acid, double-stranded nucleic acid, polypeptide chain, cellulose fiber or any flexible linear polymer and combinations thereof, and can be natural, modified or synthetic.

50. The system of claim 12, wherein The microstructure is a microcolumn or a micro-column.

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