Nucleic acid sequencing by an enzyme translocator

Abstract

Systems, devices, and methods for nucleic acid sequencing are provided. A dielectric member having a plurality of translocating proteins attached thereto positioned between a first electrode and a second electrode creates a sensing zone that allows an electroactive molecule to interact with both electrodes to complete an electrical circuit. Each of the plurality of proteins captures a polynucleotide strand, transports the polynucleotide strand into the sensing zone, and translocates the polynucleotide strand across the sensing zone at a constant rate, one nucleotide at a time. By applying a current to the first and second electrodes and holding the first electrode at a first voltage and the second electrode at a second voltage, electron transfer via an electroactive label covalently attached to a nucleotide is enabled. The current-versus-time measurements of the first and second electrodes are detected to determine when a nucleotide having an electroactive label is within the sensing zone.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Patent Application No. 63 / 349,568, filed on June 6, 2022, which is incorporated herein by reference.

[0002] In at least one aspect, the present disclosure relates to systems, devices, and methods for nucleic acid sequencing.

[0003] Background Art Single - nucleotide resolution DNA sequencing is an important goal in biotechnology. To date, most technologies require either significant reconstruction of sequences from small reads or iterative execution to achieve fidelity.

[0004] Summary of the Invention The present disclosure relates to systems, devices, and methods for nucleic acid sequencing. The systems, devices, and methods include a dielectric member with a plurality of translocating proteins attached thereto positioned between a first electrode and a second electrode. The dielectric member positioned between the first electrode and the second electrode creates a sensing zone that allows an electroactive molecule to interact with both the first electrode and the second electrode to complete an electrical circuit. On the surface of the dielectric member, two or more proteins are immobilized. Each of the two or more proteins captures a polynucleotide strand, transports the polynucleotide strand into the sensing zone, and translocates the polynucleotide strand across the sensing zone at a constant rate, one nucleotide at a time. By applying a current to the first electrode and the second electrode and holding the first electrode at a first voltage and the second electrode at a second voltage, electron transfer via an electroactive label covalently bonded to a nucleotide is enabled. When the two or more proteins are exposed to a sample containing a polynucleotide strand, the current versus time at the first electrode and the second electrode is detected to determine when a nucleotide with an electroactive label is within the sensing zone.

[0005] In another aspect, a system for nucleic acid sequencing is provided. The system includes at least one device that includes a first electrode, a second electrode, and a dielectric member positioned between the first electrode and the second electrode. Two or more proteins are immobilized on the surface of the same dielectric member. Each of the two or more proteins captures a polynucleotide chain, transports the polynucleotide chain into a sensing zone, and translocates the polynucleotide chain across the sensing zone at a constant rate, one nucleotide at a time. A control device conducts current to the first electrode and the second electrode, holds the first electrode at a first voltage, and holds the second electrode at a second voltage to enable electron transfer through an electroactive label covalently bound to a nucleotide. The control device also directs the exposure of the two or more proteins to a sample containing a polynucleotide chain. When the two or more proteins are exposed to the sample containing the polynucleotide chain, the control device induces the detection of the current versus time of the first electrode and the second electrode to determine when a nucleotide having an electroactive label is within the sensing zone. The control device then applies at least one external parameter to the at least one device to reversibly and / or repeatedly regulate the activity of the two or more proteins.

[0006] In yet another aspect, a method for forming a device for nucleic acid sequencing is provided. The method includes providing at least one device that includes a first electrode, a second electrode, and a dielectric member positioned between the first electrode and the second electrode, configuring the dielectric member to operate as a sensing zone sized such that an electroactive molecule can interact with both the first and second electrodes to complete an electrical circuit, and immobilizing two or more proteins on the surface of the dielectric member, wherein each of the two or more proteins captures a polynucleotide chain, transports the polynucleotide chain into the sensing zone, and translocates the chain across the sensing zone at a constant rate, one nucleotide at a time.

[0007] To further understand the nature, object, and advantages of the present disclosure, reference should be made to the following detailed description read in conjunction with the following drawings, in which like reference numerals indicate like elements.

Brief Description of the Drawings

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[0009] Mode for Carrying Out the Invention If necessary, detailed embodiments of the present disclosure are disclosed herein. However, it should be understood that the disclosed embodiments are merely examples and may be embodied in various alternative forms. The figures are not necessarily to scale. Some features may be exaggerated or minimized to show details of particular components. Accordingly, the specific structural and functional details disclosed herein should not be construed as limiting, but rather as a representative basis for teaching one of ordinary skill in the art.

[0010] Unless otherwise indicated by way of example or explicitly stated otherwise, all quantities in this description indicating amounts of materials or conditions of reactions and / or use are to be understood as being modified by the term "about". The first definition of an acronym or other abbreviation applies to all subsequent uses of the same abbreviation in this specification and is applied mutatis mutandis to normal grammatical variations of the initially defined abbreviation. Further, unless explicitly stated to the contrary, measurements of properties are determined by the same technique as previously or later referenced for the same property.

[0011] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0012] Also, it should be understood that the present disclosure is not limited to the specific embodiments and methods described below because certain components and / or conditions may naturally vary. Further, the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0013] As used in this specification and the appended claims, it should also be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. For example, reference to a singular component is intended to include a plurality of components.

[0014] The terms "or" and "and" can be used interchangeably and can be understood to mean "and / or".

[0015] The term "comprising" is synonymous with "including", "having", "containing", or "characterized by". These terms are inclusive and open-ended and do not exclude additional unenumerated elements or method steps.

[0016] The phrase "consisting of" excludes elements, steps, or components not specified in the claims. When this phrase appears in a clause of the body of the claim rather than immediately following the preamble, it limits only the elements recited in that clause. Other elements are not excluded from the claim as a whole.

[0017] The phrase "consisting essentially of" limits the scope of the claim to those things that, in addition to the specified materials or steps, do not materially affect the basic and novel characteristics of the claimed subject matter.

[0018] The terms "comprising", "consisting of", and "consisting essentially of" can be used interchangeably. When one of these three terms is used, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

[0019] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably in this disclosure. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" are to be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides as applicable to the described embodiments. A polynucleotide may contain one or more modified nucleotides such as methylated nucleotides and nucleotide analogs. Where present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0020] The term "sequence identity" or "identity", when measured by a sequence comparison algorithm or by visual inspection, refers to the specified percentage of residues in two nucleic acid or amino acid sequences that are identical when aligned for maximum correspondence over a specified comparison window. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity.

[0021] The term "comparison window" refers to a segment of at least about 20 contiguous positions after two arrays are optimally aligned, where the arrays can be compared to a reference array at the same number of contiguous positions. In one improvement, the comparison window is 15 - 30 contiguous positions after two arrays are optimally aligned, where the arrays can be compared to a reference array at the same number of contiguous positions. In another improvement, the comparison window is typically about 50 - about 200 contiguous positions after two arrays are optimally aligned, where the arrays can be compared to a reference array at the same number of contiguous positions.

[0022] The terms "complementary" or "complement" refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by traditional Watson - Crick or other non - traditional types. The percentage of complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson - Crick base pairing) with a second nucleic acid sequence (e.g., 4 out of 6, 5 out of 6, and 6 out of 6 are 66.67%, 83.33%, and 100% complementary, respectively). "Fully complementary" means that all consecutive residues of a nucleic acid sequence hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is at least 40%, 50%, 60%, 62.5%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%, or a percentage in between, over regions of 4, 5, 6, 7, and 8 nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0023] As used herein, the terms "translocator", "translocating protein", "enzyme", and "protein" refer to peptides, oligopeptides, polypeptides, gene products, expression products, or proteins that can translocate a polynucleotide chain. Examples of proteins that can translocate a polynucleotide chain include DNA polymerase, RNA polymerase, ribosome, single-stranded binding protein, topoisomerase, helicase, nuclease, exonuclease, endonuclease, zinc finger nuclease, RNA-guided DNA endonuclease, transcription activator-like effector nuclease, CRISPR protein, and combinations thereof.

[0024] Unless expressly stated otherwise, all R groups (e.g., R i (wherein i is an integer)) are hydrogen, alkyl, lower alkyl, C 1-6 alkyl, C 6-10 aryl, C 6-10 heteroaryl, -NO2, -NH2, -N(R’R’’)2, -N(R’R’’R’’’)3 + L - , Cl, F, Br, -CF3, -CCl3, -CN, -SO3H, -PO3H2, -COOH, -CO2R’, -COR’, -CHO, -OH, -OR’, -O - M + , -SO3 - M + , -PO3 - M + , -COO - M + , -CF2H, -CF2R’, -CFH2, and -CFR’R’’ (wherein R’, R’’, and R’’’ are C 1-10 alkyl or C 6-18 aryl groups). A single letter (e.g., "n" or "o") is 1, 2, 3, 4, or 5. In the compounds disclosed herein, a CH bond is alkyl, lower alkyl, C 1-6 alkyl, C 6-10 aryl, C 6-10heteroaryl, -NO2, -NH2, -N(R’R’’)2, -N(R’R’’R’’’)3 + L - , Cl, F, Br, -CF3, -CCl3, -CN, -SO3H, -PO3H2, -COOH, -CO2R’, -COR’, -CHO, -OH, -OR’, -O - M + , -SO3 - M + , -PO3 - M + , -COO - M + , -CF2H, -CF2R’, -CFH2, and -CFR’R’’ (wherein R’, R’’, and R’’’ are C 1-10 alkyl or C 6-18 aryl groups). The representation of a positively charged moiety or structure means that one or more negative counterions are present to balance the charge, and similarly, the representation of a negatively charged moiety or structure means that one or more positive counterions are present to balance the charge. Percent, “parts of,” and ratio values are by weight. The term “polymer” includes “oligomer,” “copolymer,” “terpolymer,” etc. The molecular weight provided for any polymer refers to the weight average molecular weight unless otherwise indicated. The description of a group or class of materials suitable or preferred for a given purpose related to the present invention means that any two or more mixtures of the members of the group or class are equally suitable or preferred. The description of a component in chemical terms refers to the component upon addition to any combination specified herein and does not necessarily exclude chemical interactions between the components of the mixture after being mixed. The first definition of an acronym or other abbreviation applies to all subsequent uses of the same abbreviation in this specification and is applied mutatis mutandis to the normal grammatical variations of the initially defined abbreviation. Further, unless explicitly stated otherwise, the measurement of a property is determined by the same technique as that previously or later referenced for the same property.

[0025] As used herein, the term "alkyl" means, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and arylenyl groups, C 1-20 , straight-chain, branched, cyclic, saturated or at least partially, optionally completely unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains. "Lower alkyl" refers to alkyl groups having from 1 to about 8 carbon atoms (i.e., C 1-8 alkyl), for example, alkyl groups having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. Lower alkyl can also refer to a range between the two numbers of carbon atoms listed above. "Higher alkyl" refers to alkyl groups having from about 10 to about 20 carbon atoms, for example, alkyl groups having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Higher alkyl can also refer to a range between the two numbers of carbon atoms listed above.

[0026] As used herein, the term "aryl" means an aromatic substituent that can be a single aromatic ring, or a plurality of aromatic rings that are fused to each other, covalently bonded, or linked by a common group such as a methylene or ethylene moiety, but not limited thereto. The common linking group can also be carbonyl as in the case of benzophenone, or oxygen as in the case of diphenyl ether. Examples of aryl include, but are not limited to, phenyl, naphthyl, biphenyl, and diphenyl ether. The aryl group includes a heteroaryl group, and one or more aromatic rings contain heteroatoms (e.g., N, O, S, or Se). Exemplary heteroaryl groups include, but are not limited to, furanyl, pyridyl, pyrimidinyl, imidazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinolinyl, isoquinolinyl, thiophenyl, etc. The aryl group may be optionally substituted with one or more aryl group substituents that may be the same or different (a "substituted aryl"), and the "aryl group substituent" includes alkyl (saturated or unsaturated), substituted alkyl (e.g., haloalkyl and perhaloalkyl, e.g., -CF3, but not limited thereto), cycloalkyl, aryl, substituted aryl, aralkyl, halo, nitro, hydroxyl, acyl, carboxyl, alkoxyl (e.g., methoxy), aryloxyl, aralkyloxyl, thioalkyl, thioaryl, thioaralkyl, amino (e.g., aminoalkyl, aminodialkyl, aminoaryl, etc.), sulfonyl, and sulfinyl.

[0027] It should also be understood that an integer range explicitly includes all intervening integers. For example, the integer range 1 to 10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range from 1 to 100 includes 1, 2, 3, 4....97, 98, 99, 100. Similarly, when any range is required, the intervening numbers, which are the increment obtained by dividing the difference between the upper and lower limits by 10, can be regarded as alternative upper or lower limits. For example, when the range is from 1.1 to 2.1, the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as the lower or upper limit. In the specific examples described herein, the concentration, temperature, and reaction conditions (e.g., pressure, pH, etc.) can be carried out by rounding to three significant figures plus or minus 50 percent of the indicated value. In one improvement, the concentration, temperature, and reaction conditions (e.g., pressure, pH, etc.) can be carried out by rounding to three significant figures plus or minus 30 percent of the indicated value, rounded to the significant figures of the value provided in the example. In another improvement, the concentration, temperature, and reaction conditions (e.g., pH, etc.) can be carried out by rounding to three significant figures plus or minus 10 percent of the indicated value, rounded to the significant figures of the value provided in the example.

[0028] In the examples described herein, the concentration, temperature, and reaction conditions (e.g., pressure, pH, flow rate, etc.) can be carried out by rounding or truncating plus or minus 50 percent of the indicated value to two significant figures of the value provided in the example. In one improvement, the concentration, temperature, and reaction conditions (e.g., pressure, pH, flow rate, etc.) can be carried out by rounding or truncating plus or minus 30 percent of the indicated value to two significant figures of the value provided in the example. In another improvement, the concentration, temperature, and reaction conditions (e.g., pressure, pH, flow rate, etc.) can be carried out by rounding or truncating plus or minus 10 percent of the indicated value to two significant figures of the value provided in the example.

[0029] Throughout this application, when publications are referenced, the disclosures of these publications are hereby incorporated by reference in their entirety to more fully describe the state of the art to which this invention pertains.

[0030] The present disclosure discloses a device capable of reading long reads at single-base pair resolution. The present disclosure also incorporates, as methods of introducing DNA into a probe device, the addition of a translocating protein such as a biological polymerase, as described in U.S. patent application Ser. No. 16 / 009,766, filed Jun. 15, 2018, and U.S. Provisional Application No. 62 / 581,366, filed Nov. 3, 2017, both of which are hereby incorporated by reference in their entirety. The advantages of this modality are that the translocating protein acts as a controlled localization site for introducing DNA into the sensing zone while providing a controlled translocation rate within the sensing zone, which are two parameters to be controlled for single-base resolution sequencing.

[0031] To achieve the goal of introducing a polynucleotide strand such as DNA to the sensing zone, controlling the translocation rate, and reducing the number of fabrication steps required to produce the working device, a translocating protein such as DNA polymerase is bound to the dielectric gap between the oxidation electrode and the reduction electrode or to the surface of the member.

[0032] Figure 1A shows a system 10 for nucleic acid sequencing. System 10 includes at least one device 12 that includes an oxidation electrode 18, a reduction electrode 20, and a dielectric member 16 positioned between the oxidation electrode 18 and the reduction electrode 20. Characteristically, the dielectric member 16 separates the reduction electrode 20 and the oxidation electrode 16 by a first distance 28 of up to 10 nm. Protein 22 is attached 24 to the surface 25 of the dielectric. Protein 22 can translocate a polynucleotide strand that can receive a modified nucleotide having a redox label attached to the nucleotide or a redox label covalently bonded to the nucleoside base of the modified nucleotide. As is well known, nucleotides include nucleoside bases, which are sometimes referred to as nucleic acid bases. In this context, the term redox label includes a fully functional redox label or moiety that can react to form a functional redox label. Also, the term "covalently bonded to the nucleoside base of the modified nucleotide" means that the moiety containing the redox label is covalently bonded to the nucleotide. In at least one aspect, the modified nucleotide is modified for the redox label attached thereto. Attachment 24 of protein 22 is such that a modified nucleotide having a redox label covalently bonded to the nucleoside base of the polynucleotide strand passes within a second distance of up to 10 nm from the surface of the dielectric member during translocation. The oxidation and reduction electrodes 18, 20 create an electric field that extends to a reaction region where translocation of the polynucleotide strand by the protein occurs. Advantageously, the spatial dimensions allow for rapid electron transfer (i.e., substantially simultaneously) to the redox label from the reduction electrode to the oxidation electrode when the modified nucleotide having a redox label covalently bonded to the nucleoside base of the modified nucleotide is located in the reaction region. Mover, the spatial dimensions are such that diffusion is not an important factor in electron transport.

[0033] FIG. 1A also shows a device 12 including an electrode pair type device 14 that includes a dielectric member 16 positioned between an oxidation bias electrode or oxidation electrode 18 and a reduction bias electrode or reduction electrode 20. U.S. Patent Application No. 16 / 009,766 and U.S. Provisional Application No. 62 / 581,366 disclose exemplary embodiments of an electrode pair type device 14 and a method of fabricating the electrode pair type device 14. Both U.S. Patent Application No. 16 / 009,766 and U.S. Provisional Application No. 62 / 581,366 disclose methods of DNA sequencing using redox labels and the shuttle principle. Briefly, the shuttle detection mechanism includes two electrodes separated by a dielectric of nanoscale thickness. The electrodes are held at oxidation and reduction potentials to enable reversible electrochemical reactions of redox molecules. The small space between the two electrodes is called the sensing zone, which is small enough for redox molecules to interact with both electrodes to complete an electrical circuit. While the redox molecules are present in the sensing zone, electrons can "shuttle" between the reduction electrode and the oxidation electrode, generating an amplified current signal that is much higher than the signal expected from a single electron transfer event. This mechanism is different from a nanogap device in which redox molecules must diffuse back and forth between the electrodes to generate a measurable electrical signal.

[0034] Dielectric members 16 of various embodiments include materials having a dielectric constant such that fluctuations in the tunnel current between the oxidation electrode 18 and the reduction electrode 20 are smaller than changes in the current flow resulting from electron transfer from the reduction electrode 20 to the redox label and the oxidation electrode 18. Examples of materials include hafnium and zirconium silicate, metal oxides or nitrides such as aluminum oxide, titanium dioxide, hafnium oxide, zirconium oxide, silicon oxide, silicon nitride, and hexagonal boron nitride.

[0035] In various embodiments, the dielectric member 16 separates the reduction electrode 20 and the oxidation electrode 18 by a first distance 28 of up to 10 nm. In various embodiments, the dielectric member 16 has a width 28 in the range of 1 nm to 10 nm, preferably in the range of 1 nm to 4 nm, between the oxidation electrode 18 and the reduction electrode 20. In various embodiments, the width 28 of the dielectric member 16 between the oxidation electrode 18 and the reduction electrode 20 is 0.5 nm, 1 nm, 1.25 nm, 1.5 nm, 1.75 nm, 2 nm, 2.25 nm, 2.5 nm, 2.75 nm, 3 nm, 3.25 nm, 3.5 nm, 3.75 nm, 4 nm, 4.25 nm, 4.5 nm, 4.75 nm, 5 nm, 5.25 nm, 5.5 nm, 5.75 nm, 6 nm, 6.25 nm, 6.5 nm, 6.75 nm, 7 nm, 7.25 nm, 7.5 nm, 7.75 nm, 8 nm, 8.25 nm, 8.5 nm, 8.75 nm, 9 nm, 9.25 nm, 9.5 nm, 9.75 nm, or 10 nm. In various embodiments, the width 28 of the dielectric member 16 is in the range between any two of the above-specified widths.

[0036] One parameter is the cross-sectional area of the dielectric member 16 defined by the thickness 35 and the lengths 31, 33 between the oxidation electrode 18 and the reduction electrode 20. The cross-sectional area of the dielectric member 16 is preferably small enough to allow for an electron shuttle while providing sufficient insulation between the electrodes to avoid a short circuit.

[0037] In various embodiments shown in FIG. 1B, the dielectric member 16 has a thickness 35 in the range of 5 nm to 5000 nm, preferably 10 nm to 1000 nm. In various embodiments, the thickness 35 of the dielectric member 16 is 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm. In various embodiments, the thickness 35 of the dielectric member 16 is in the range between any two of the above-specified thicknesses.

[0038] In various embodiments, the oxidation electrode 18 has a width 30 that contacts a sample or solution in the range of 5 nm to 5000 nm, preferably 10 nm to 1000 nm. In various embodiments, the width 30 of the oxidation electrode 18 that contacts the sample or solution is 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm. In various embodiments, the width 30 of the oxidation electrode 18 that contacts the sample or solution is in the range between any two of the above-specified widths.

[0039] Various embodiments are shown in FIG. 1C, and the oxidation electrode 18 has a length 31 that contacts a sample or solution in the range of 10 nm to 10,000 nm, preferably 50 nm to 5,000 nm. In various embodiments, the length 31 of the oxidation electrode 18 that contacts the sample or solution is 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, 5000 nm, 6000 nm, 7000 nm, 8000 nm, 9000 nm, or 10,000 nm. In various embodiments, the length 31 of the oxidation electrode 18 that contacts the sample or solution is in the range between any two of the specified lengths above.

[0040] In various embodiments, the reduction electrode 20 has a width 32 that contacts a sample or solution in the range of 5 nm to 5,000 nm, preferably 10 nm to 1,000 nm. In various embodiments, the width 32 of the reduction electrode 20 that contacts the sample or solution is 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm. In various embodiments, the width 32 of the reduction electrode 20 that contacts the sample or solution is in the range between any two of the specified widths above.

[0041] Various embodiments are shown in FIG. 1C, and the reduction electrode 20 has a length 33 that contacts a sample or solution in the range of 10 nm to 10,000 nm, preferably 50 nm to 5000 nm. In various embodiments, the length 33 of the reduction electrode 20 that contacts the sample or solution is 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, 5000 nm, 6000 nm, 7000 nm, 8000 nm, 9000 nm or 10,000 nm. In various embodiments, the length 33 of the reduction electrode 20 that contacts the sample or solution is in the range between any two of the specified lengths above.

[0042] In various embodiments shown in FIGS. 1D and 1E, the overlap 41 between the oxidation electrode 18 and the reduction electrode 20 has a length 45 in the range of 10 nm to 10,000 nm, preferably 50 nm to 5000 nm, and a width 43 in the range of 1 nm to 10 nm, preferably 1 nm to 4 nm. The overlap 41 between the oxidation electrode 18 and the reduction electrode 20 can be understood as the superposition of the electric fields from the oxidation electrode 18 and the reduction electrode 20.

[0043] The length 45 of the overlap 41 of various embodiments is 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, 5000 nm, 6000 nm, 7000 nm, 8000 nm, 9000 nm, or 10000 nm. In various embodiments, the length 45 is in the range between any two of the specified lengths above.

[0044] The width 43 of the overlap 41 of various embodiments and the width 28 of the dielectric member 16 between the oxidation electrode 18 and the reduction electrode 20 are 0.5 nm, 1 nm, 1.25 nm, 1.5 nm, 1.75 nm, 2 nm, 2.25 nm, 2.5 nm, 2.75 nm, 3 nm, 3.25 nm, 3.5 nm, 3.75 nm, 4 nm, 4.25 nm, 4.5 nm, 4.75 nm, 5 nm, 5.25 nm, 5.5 nm, 5.75 nm, 6 nm, 6.25 nm, 6.5 nm, 6.75 nm, 7 nm, 7.25 nm, 7.5 nm, 7.75 nm, 8 nm, 8.25 nm, 8.5 nm, 8.75 nm, 9 nm, 9.25 nm, 9.5 nm, 9.75 nm, or 10 nm. In various embodiments, the width 43 is in the range between any two of the specified widths above.

[0045] In various embodiments, the oxidation electrode 18 or the reduction electrode 20 is a planar electrode. The oxidation electrode 18 or the reduction electrode 20 of various embodiments includes materials such as titanium nitride, palladium, or platinum. Examples of electrodes for use in various embodiments of the system and device are disclosed in U.S. Patent Application Publication No. 2017 / 0370870, which is hereby incorporated by reference in its entirety.

[0046] Translocating protein 22 is a protein that can bind to polynucleotide strands such as double-stranded or single-stranded DNA and RNA, and can translocate or shuttle the polynucleotide strand by the protein. Examples of translocating proteins include DNA polymerases such as Taq polymerase, RNA polymerases such as T7 RNA polymerase, ribosomes, single-strand binding proteins, topoisomerases, helicases, nucleases, exonucleases, endonucleases, zinc finger nucleases, RNA-guided DNA endonucleases, transcription activator-like effector nucleases, and CRISPR proteins.

[0047] For example, other potential enzymes for holding and scanning the DNA strand include nucleases such as exonucleases, endonucleases, deoxyribonucleases, and ribonucleases, helicase enzymes, and CRISPR proteins. Examples of CRISPR proteins are CRISPR-Cas type and CRISPR-related proteins including, but not limited to, Cas9 and Csf1. In the case of CRISPR-related enzymes, devices of various embodiments include using a gRNA target as a guide designed not to recognize portions of the DNA strand being sequenced. The enzyme controls the translocation and reading of the entire target DNA within the sensing zone.

[0048] In various embodiments, Protein 22 attaches to the surface 25 of the dielectric member 16 such that a modified nucleotide having a redox label covalently bound to the nucleoside base of the modified nucleotide of the polynucleotide chain passes up to 10 nm from the surface of the dielectric member. In various embodiments, the protein has a modified nucleotide having a redox label covalently bound to the nucleoside base of the modified nucleotide of the polynucleotide chain pass 0 nm, 0.25 nm, 0.5 nm, 0.75 nm, 1 nm, 1.25 nm, 1.5 nm, 1.75 nm, 2 nm, 2.25 nm, 2.5 nm, 2.75 nm, 3 nm, 3.25 nm, 3.5 nm, 3.75 nm, 4 nm, 4.25 nm, 4.5 nm, 4.75 nm, 5 nm, 5.25 nm, 5.5 nm, 5.75 nm, 6 nm, 6.25 nm, 6.5 nm, 6.75 nm, 7 nm, 7.25 nm, 7.5 nm, 7.75 nm, 8 nm, 8.25 nm, 8.5 nm, 8.75 nm, 9 nm, 9.25 nm, 9.5 nm, 9.75 nm or 10 nm from the surface of the dielectric member. In various embodiments, the distance that a modified nucleotide having a redox label covalently bound to the nucleoside base of the modified nucleotide of the polynucleotide chain passes from the surface of the dielectric member is in the range between any two of the above-specified distances.

[0049] Figures 2A, 2B, 2C, 3 and 4 show the device 12 incorporated in different structures.

[0050] Figure 2A shows an arrangement 100 in which the device 12 or the protein 22 is exposed to an opening 101 into which a sample can be added, the device 12 including electrodes 18, 20, and a dielectric member 16 to which the translocate protein 22 is attached. Figure 2B shows a device 12 including electrodes 18, 20, and a dielectric member 16 to which the translocate protein 22 is attached, incorporated within a wall 102 of a channel (nanoscale channel), the device 12 or the protein 22 being exposed to a channel 103 into which a sample can be added. Proteins such as polymerase that are attached onto a dielectric member between planar electrodes do not require a nanoscale channel, but may be within a channel or an open solution as shown in Figure 14 (Open and Channel).

[0051] Figure 2C shows a device 12 including electrodes 18, 20, and a dielectric member 16 to which the translocate protein 22 is attached, as a floor 104 of a well 104. The device 12 or the protein 22 is exposed to a channel 105 into which a sample can be added.

[0052] Figure 3 shows a plurality of devices 12 as part of a well 106. The devices 12 include electrodes 18, 20, and a dielectric member 16 to which the translocate protein 22 is attached. As shown in Figure 3, the well 106 has opposing sidewalls 108, 110 attached to a floor 112 that defines a channel 114. The device 12 can be incorporated into the sidewalls 108, 110 or the floor 112 such that the protein 22 is positioned within the channel 114. For example, as shown in Figure 3, an alternative fabrication method is possible in which the structure is formed at the edge of the well.

[0053] Figure 4 shows sidewalls 116, 118 that define a channel 120 that is smaller in size compared to the channel 114, the device 12 being incorporable into the sidewall 116 such that the protein 22 is positioned within the channel 114. The device 12 includes electrodes 18, 20, and a dielectric member 16 to which the translocate protein 22 is attached.

[0054] The method of fabricating the electrode pair form device 14 of the device 12 shown in FIGS. 2A, 2B, 2C, 3, and 4 is described in U.S. Patent Application No. 16 / 009,766 and U.S. Provisional Application No. 62 / 581,366 with modifications.

[0055] Figure 5 shows a schematic diagram of a process for fabricating the device 12 of various embodiments. In step 34, the surface 25 of the dielectric member 16 is modified 26 to include the adhesive 24. In steps 36 and 38, the translocate protein 22 is attached to the dielectric member 16 via attachment 40 to the adhesive 24. The conjugation of the translocate protein 22 reacts with the dielectric member at one end, for example, silane chemistry or organophosphoric acid chemistry, and reacts with a biomolecule at the other end, for example, carboxyl, aldehyde, sulfonic acid, isothiocyanate, NHS ester, epoxide or carbodiimide chemistry, and can be controlled by using a bifunctional coupling agent 24. It should be understood that steps 34, 36, and 38 (e.g., 34>>36, 38) can be performed sequentially, simultaneously, or in a different order (e.g., 36, 38>>34). The chemistry is preferably selective for the dielectric material (e.g., aluminum oxide) with respect to the metal electrodes such that covalent bonding occurs on the dielectric member between the electrodes and does not occur on the metal electrodes. In one example, the bifunctional coupling agent is 3-aminopropyltriethoxysilane. An example of attachment via silane chemistry is disclosed by Sin, Eun Jung, et al. “Surface modification of aluminum oxide for biosensing application.” Biomedical Engineering: Applications, Basis and Communications 24.02 (2012): 111-116, which is incorporated by reference in its entirety. An example of attachment via organophosphoric acid chemistry is disclosed by Mutin, P. Hubert, et al. “Selective surface modification of SiO2-TiO2 supports with phosphonic acids.” Chemistry of materials 16.26 (2004): 5670-5675, which is incorporated by reference in its entirety. Alternatively, the translocate protein can be physically adsorbed onto the dielectric layer rather than covalently bonded.In one example, as a result, a polymerase covalently bound between two electrodes is obtained, where the binding pocket allows the internal chemistry to interact with the electrodes. Thus, when DNA is being replicated, when a redox-modified base enters the enzyme active site, it begins to undergo electrochemical oxidation and reduction reactions with the electrodes. This enables an electron shuttle that is used to detect the presence of modified bases used in sequencing.

[0056] Figures 6 and 7 show systems 1020, 1040 that include arrays 42, 44 of device 12. In various embodiments, the system includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 2500, 5000, 10000, or 100000 devices. In various embodiments, the number of devices is in the range between any two of the above-specified numbers of devices. In one improvement, the proteins in each of the arrays are uniformly distributed.

[0057] Figures 8A, 8B, 9A, and 9B show that protein 22 is at least partially aligned on the surface of dielectric member 16. In this regard, alignment means that the orientation of the principal axes of inertia of each protein in a plurality of devices 12 is not random.

[0058] Figure 8A shows a system that includes two devices 12 to which proteins 22, 22' are attached. As shown in Figure 8A, proteins 22 and 22' are aligned. In Figure 8A, for simplicity, only the corresponding principal axes of inertia 51 and 54 are drawn and are shown to be aligned between the proteins. Figure 8B shows proteins 22 and 22' that are oriented such that the corresponding principal axes of inertia 51, 54 can be offset from each other by angle A2.

[0059] Figures 9A and 9B show a general case where proteins 22 and 22' are slightly misaligned. Protein 22 defines associated principal axes of inertia l1, l2, l3, and protein 22' defines associated principal axes of inertia l'1, l'2, l'3. The first principal axes of inertia l1, l'1 may be offset from each other by a maximum angle A1, the second principal axes of inertia l2, l'2 may be offset from each other by a maximum angle A2, and the third principal axes of inertia l3, l'3 may be offset from each other by a maximum angle A3, and each of the angles A1, A2, and A3 is at most 60 degrees. Since protein 22 is at least substantially uniformly oriented on the surface of dielectric member 16, the offsets of the corresponding principal axes of inertia between the proteins are relatively small angles with respect to each other. In one improvement, A1, A2, and A3 are at most 45 degrees. In a further improvement, A1, A2, and A3 are at most 0°, 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 11°, 12°, 13°, 14°, 15°, 16°, 17°, 18°, 19°, 20°, 21°, 22°, 23°, 24°, 25°, 26°, 27°, 28°, 29°, 30°, 31°, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41°, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, 50°, 51°, 52°, 53°, 54°, 55°, 56°, 57°, 58°, 59°, or 60°. In various embodiments, the offset is in the range between any two of the specified angles above.

[0060] Figures 10 and 11 show protein 22 at least partially aligned on the surface of dielectric member 16. In this regard, alignment means that the proteins are uniformly distributed over a predetermined region including a plurality of devices 12.

[0061] Figure 10 shows a system 1100 including three devices 12. As shown in FIG. 8, protein 22 is attached to dielectric member 16 at a position such that the reaction region where translocation 57 occurs within protein 22, or the sensing zone 57 of oxidation electrode 18 and reduction electrode 20, are in the same position.

[0062] FIG. 11 shows a system 1120 that includes three devices 12. As shown in FIG. 9, the protein 22 is attached at different positions such that the translocation 57, 57', 57'' occurs within the reaction region of the protein 22, or the sensing zones 57, 57', 57'' of the oxidation electrode 18 and the reduction electrode 20 are within the zone 58. In various embodiments, the proteins are attached to the dielectric member such that the reaction regions or sensing zones of the proteins are at distances of 0 nm, 0.1 nm, 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, and 2 nm from each other. In various embodiments, the distance is in the range between any two of the specified distances above.

[0063] FIG. 12 shows a schematic diagram of the fabrication of systems 1020, 1040, 1100, 1120 including a method for forming a nucleic acid sequencing device. The method includes, prior to step 60, providing a device 14 including an oxidation electrode 18, a reduction electrode 20, and a dielectric member 16. Characteristically, the dielectric member 16 separates the reduction electrode 20 and the oxidation electrode 20 by a first distance of up to 10 nm. The method includes a second step 62 of creating an electric field 66 by the oxidation electrode 18, the reduction electrode 16, or both. The method also includes a third step 64 of attaching 24 a protein 22 to the surface 25 of the dielectric member 16. The protein 22 is such that a modified nucleotide having a redox label covalently bonded to the nucleoside base of the modified nucleotide of the polynucleotide chain can translocate a polynucleotide chain having a nucleotide modified with a redox label such that it passes from the surface of the dielectric member by a second distance of up to 10 nm during translocation, or a modified nucleotide having a redox label covalently bonded to the nucleoside base of the modified nucleotide can be received on the surface of the dielectric member. In step 64, a translocation protein 22 such as a polymerase is guided to the dielectric member 16 by the electric field 66 and induced by the electric field 66 to an at least substantially uniform orientation. In different examples, the voltage can be selected such that the polymerases attract the charged polymerases with symmetric forces so that the polymerases bind between the electrodes. Alternatively, a transverse electric field can be generated to control the orientation of the polymerase molecules such that a relatively uniform orientation can result in improved sensor performance. An example of a transverse electric field for controlling the orientation of a protein is disclosed in Emaminejad, Sam, et al. “Tunable control of antibody immobilization using electric field.” Proceedings of the National Academy of Sciences 112.7 (2015): 1995-1999, which is hereby incorporated by reference in its entirety.

[0064] For example, during the polymerase immobilization process, an electric field created by electrodes can be used to guide biomolecules to the dielectric layer and induce a uniform orientation on the surface. For example, the voltage can be selected such that the electrodes attract the charged polymerase with symmetric forces so that the polymerase binds between the electrodes. If it is necessary to selectively adsorb the polymerase onto the dielectric layer, the voltage on the electrodes can be set to generate surface charges that are unfavorable for the attachment of the polymerase to the electrodes (adsorption decreases when the surface charge matches the isoelectric point of the polymerase). Alternatively, a uniform orientation can result in improved sensor performance (see Emaminejad et al.), so a transverse electric field can be generated to control the orientation of the polymerase molecules.

[0065] Figures 13, 14, 15, and 16 illustrate a method for nucleic acid sequencing. The method includes a first step of providing at least one device including an oxidation electrode 18, a reduction electrode 20, a dielectric member 16, and a protein 22 attached 24 to a surface 25 of the dielectric member 16. Characteristically, the dielectric member 16 separates the reduction electrode 20 and the oxidation electrode 18 by a first distance of up to 10 nm. The protein 22 can translocate a polynucleotide strand that has modified nucleotides with redox labels or can receive modified nucleotides having a redox label covalently bonded to the nucleoside base of the modified nucleotide. The attachment 64 of the protein 22 is such that a modified nucleotide having a redox label covalently bonded to the nucleoside base of the polynucleotide strand passes within a second distance of up to 10 nm from the surface 25 of the dielectric member 16 during translocation. The method includes a third step of applying a current to the oxidation electrode 18 and the reduction electrode 20, the oxidation electrode 18 and the reduction electrode 20 creating an electric field that extends to a reaction region where translocation of the polynucleotide strand by the protein 22 occurs. The method includes a third step of exposing the protein 22 to a sample containing the polynucleotide strand, enabling the polynucleotide strand to be translocated by the protein 22. The method includes a fourth step of detecting a change in the flow of current at the oxidation electrode 18 and the reduction electrode 20. The change identifies the electron transfer from the reduction electrode 20 to the redox label and the oxidation electrode 18 when a modified nucleotide having a redox label covalently bonded to the nucleoside base of the polynucleotide strand is in the reaction region.

[0066] Figure 13 shows a device 12 that includes a dielectric member 16 between an oxidation electrode 18 and a reduction electrode 20, with DNA polymerases 22, 68 attached to the dielectric member 16 at 24. When current is conducted to electrodes 18, 20, an electric field 66 is created by the oxidation electrode 18 and the reduction electrode 20 and is directed to a sensing zone 70 that includes the active site 72 of DNA polymerase 68. Using a primer 74, DNA polymerase 68 generates a complementary strand 76 to the base strand 78 by incorporating dNTPs 82 that are modified to include free deoxynucleotides (dNTPs) 80 and redox labels 83. When a modified dNTP 82 or a redox label enters the active site 72 and the sensing zone 70 during DNA replication, an electron transfer 84 from the reduction electrode 20 to the redox label 83 and to the oxidation electrode 18 occurs. In the embodiment shown in Figure 13, the redox label 83 enters or is adjacent to the active site 72 during DNA replication by the polymerase 68, and a base-specific signal is provided each time a modified nucleotide having a redox label covalently bonded to the nucleoside base of the modified nucleotide 82 is incorporated. In different examples, the electron transfer can occur without the diffusion of the redox label 83. Since the incorporation rate of the polymerase can be determined, bases can be assigned as a function of signal versus time. The device operates such that one base at a time is redox-modified during the replication process. Multiple devices can be run in parallel to simultaneously achieve the detection of different bases. If there are redox species in the solution, redox label species that freely diffuse in the solution can also interact within the electrodes. However, the time constants of molecules that freely diffuse through the sensing zone are different from those of molecules that are constrained within the sensing zone during incorporation. Thus, looking at the frequency domain of the signal allows the distinction between diffusion and translocation signals. In different examples, the polymerase 68 is immobilized on the surface of the dielectric member 16 at the nm scale between the two electrodes 18, 20. The polymerase 68 can bind to DNA 78 and primer 74 and initiate the incorporation of nucleotides 80, 82 via a polymerase chain reaction. The sensing zone 70 is within the overlap between the oxidation electrode 18 and the reduction electrode 20.

[0067] In various embodiments, the electron transfer 84 from the reduction electrode 20 to the redox label 83 and the oxidation electrode 18 occurs at a rate (i.e., electron transfer rate) in the range of #x10 6 s -1 ~#x10 12 s -1 where # is any value in the range of 1 to 10. In various embodiments, the electron transfer rate is #x10 6 s -1 、#x10 7 s -1 、#x10 8 s -1 、#x10 9 s -1 、#x10 10 s -1 、#x10 11 s -1 、#x10 12 s -1 where # is any value in the range of 1 to 10. In various embodiments, the electron transfer rate is a rate selected between any two of the above-specified rates. For example, the electron transfer rate from the reduction electrode 20 to the redox label 83 and the oxidation electrode 18 occurs at a rate of #x10 6 s -1 where # is any value in the range of 1 to 10.

[0068] In various embodiments, the voltages of the oxidation electrode 18 and the reduction electrode 20 in the guiding step are different from each other.

[0069] In an alternative embodiment, the front end of the electronic device can be arranged in a fully differential manner. When a current is flowing through one electrode of the front end, a current having the same amplitude but a different polarity is flowing through the second input electrode of the front end. Thereby, it is avoided that the disturbance applied to both electrodes is transmitted through the signal path. Examples of this embodiment are disclosed and described in U.S. Patent Application No. 16 / 009,766 and U.S. Provisional Application No. 62 / 581,366.

[0070] In another embodiment, a high-pass characteristic is used at the first stage of the front end, thereby avoiding differential DC currents (from tunneling or current flowing through polymerase) that would overload the signal path of the front end. This avoids the signal being pushed to the limits of the measurement range due to "parasitic" DC currents. The change in current from the base for detection is transmitted through the high-pass and processed within the electronic signal chain.

[0071] The redox labels 83 of various embodiments are compounds that can be oxidized by the oxidation electrode 18 and reduced by the reduction electrode 20. Examples of redox labels include ferrocene (cyclopenta-1,3-diene; iron(2+)) and its derivatives, anthraquinone (anthracene-9,10-dione), methylene blue ([7-(dimethylamino)phenothiazine-3-ylidene]-dimethylazanium; chloride), as well as phenothiazine (10H-phenothiazine), osmium and ruthenium complexes, tetrathiafulvalene, aminophenol, nitrophenol, erythrosin B, ATTO MB2, and the like. The redox species undergo reversible redox reactions under the applied potential to enable the shuttle detection principle.

[0072] In various embodiments, the method and system include dNTPs 82 modified with different redox labels, each redox label having a different redox potential. In an example, the method includes two, three, or four nucleotide dNTPs having different redox labels. For example, adenine, thymine, or uracil may be modified to include a redox label, and cytosine or guanine may be modified to include a different redox label. Examples of methods for replicating a DNA strand and incorporating redox-modified nucleotides are disclosed in U.S. Patent Application No. 16 / 009,766 and U.S. Provisional Application No. 62 / 581,366.

[0073] In various embodiments, a modified nucleotide having a redox label covalently attached to the nucleoside base of the modified nucleotide 82 has the following formula: [Chemical formula] (wherein X is [Chemical formula] and [Chemical formula] is a single bond, double bond, or triple bond, Lk is absent or is a hydrocarbon-containing linking group including alkyl, aryl, heteroalkyl, heteroaryl, cycloalkyl, or a heteroatom-containing ring system, R1 is H or OH, R2 is a redox label) has.

[0074] Examples of modified nucleotides having a redox label covalently attached to the nucleoside base of modified nucleotide 82 include the following formula: [Chemical formula] compounds having.

[0075] Examples of modified nucleotides having a precursor for a redox label covalently attached to the nucleoside base of modified nucleotide 82 include the following formula: [Chemical formula] [Chemical formula] [Chemical formula] compounds having, m is 1 to 12, n is 1 to 100, o is 3 to 12.

[0076] FIG. 14 is similar to FIG. 13, except that the base strand 78’ contains a redox-modified nucleotide 86 to which a redox label is attached. Using primer 74, DNA polymerase 22, 68 incorporates free dNTP 80 to generate a complementary strand 76’ to the base strand 78’. When the modified nucleotide 86 or the redox label 83 of the base strand 78’ enters the active site 70 or the sensing zone 72 during DNA replication, electron transfer occurs from the reduction electrode 20 to the redox label 83 and to the oxidation electrode 18 84, thereby changing the current flow through the electrodes 18, 20 and resulting in a base-specific signal. The method of incorporation may include any DNA that already has a single strand modified with a redox incorporation species as shown in FIG. 14 and is described in U.S. Patent Application No. 16 / 009,766 and U.S. Provisional Application No. 62 / 581,366. Alternatively, unmodified DNA is used and the redox-modified base is in solution.

[0077] FIG. 15 is similar to FIGS. 13 and 14, except that a nuclease 22, 88 attached 24 to the dielectric member 16 is used instead of DNA polymerase 68. Nuclease 88 binds to a polynucleotide strand 90 having a redox-modified nucleotide 86 to which a redox label is attached. During digestion of the polynucleotide strand 90 into fragments 92, nuclease 88 translocates the polynucleotide strand 90 such that the redox-modified nucleotide 86 or the redox label 83 of the polynucleotide strand 90 enters the sensing zone 70. Then, electron transfer occurs from the reduction electrode 20 to the redox label 83 and to the oxidation electrode 18 84, thereby changing the current flow through the electrodes 18, 20 and resulting in a base-specific signal.

[0078] FIG. 16 is the same as FIGS. 13, 14, and 15, except that it uses the CRISPR-associated protein 9 nuclease 22, 94 attached 24 to the dielectric member 16. A CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) containing a constant region 96 and a targeting region 98 is positioned within the CRISPR-associated protein 9 nuclease 94. The polynucleotide sequence of the targeting region 98 is such that the CRISPR-associated protein 9 nuclease 94 translocates the polynucleotide strand 90 having the redox-modified nucleotides 86, 83 without causing a double-strand break. During translocation, the modified nucleotide 86 or the redox label 83 of the polynucleotide strand 90 enters the sensing zone 70. Then, electron transfer occurs 84 from the reduction electrode 20 to the redox label 83 and to the oxidation electrode 18, thereby changing the flow of current through the electrodes 18, 20 to provide a base-specific signal.

[0079] In various embodiments, the electron transfer 84 from the reduction electrode 20 to the redox label 83 and to the oxidation electrode 18 occurs at a rate (i.e., electron transfer rate) in the range of #x10 6 s -1 ~#x10 12 s -1 where # is any value in the range of 1 to 10. In various embodiments, the electron transfer rate is #x10 6 s -1 、#x10 7 s -1 、#x10 8 s -1 、#x10 9 s -1 、#x10 10 s -1 、#x10 11 s -1 、#x10 12 s -1 and # is any value in the range of 1 to 10. In various embodiments, the electron transfer rate is a rate selected between any two of the above-specified rates. For example, the electron transfer rate from the reduction electrode 20 to the redox label 83 and to the oxidation electrode 18 is #x106 s -1 occurs at a rate of #, where # is any value in the range of 1 to 10.

[0080] In various embodiments, the voltages of the oxidation electrode 18 and the reduction electrode 20 in the guiding step are different from each other.

[0081] The redox labels 83 of various embodiments are compounds that can be oxidized by an oxidation electrode and reduced by a reduction electrode. Examples of redox labels include ferrocene (cyclopenta-1,3-diene; iron(2+)) and its derivatives, anthraquinone (anthracene-9,10-dione), methylene blue ([7-(dimethylamino)phenothiazine-3-ylidene]-dimethylazanium; chloride), and phenothiazine (10H-phenothiazine), osmium and ruthenium complexes, tetrathiafulvalene, aminophenol, nitrophenol, erythrosin B, ATTO MB2, etc. The redox species undergo a reversible redox reaction under the applied potential to enable the shuttle detection principle.

[0082] In various aspects, the method and system include nucleotides 86 modified with different redox labels having different potentials. In an example, the method includes two, three, or four nucleotides having different redox labels. For example, adenine, thymine, or uracil may be modified to include a redox label, and cytosine or guanine may be modified to include a different redox label. Examples of methods for replicating a DNA strand and incorporating redox-modified nucleotides are disclosed in U.S. Patent Application No. 16 / 009,766 and FIGS. 4 and 5 and paragraphs 0017 - 0019 of U.S. Provisional Application No. 62 / 581,366.

[0083] In various embodiments, the modified nucleotide 86 has the following formula:

Chemical formula

Chem.

Chem.

[0084] Examples of modified nucleotide 86 with a redox label attached include the following formula:

Chem.

[0085] Examples of modified nucleotide 86 with a redox label precursor attached include the following formula:

Chem.

Chem.

Chem.

[0086] Examples of methods for synthesizing dNTPs that form modified dNTP82 or redox-modified nucleotides 86 with attached redox labels are shown below.

[0087] Redox labels can be introduced directly into the target DNA by synthesizing nucleotides containing the label, and the nucleotides can be incorporated into the DNA strand during PCR (Figures 17 and 18). Alternatively, a two-step approach can be used (Figure 19): Nucleotides containing a chemical "handle" can be introduced into the DNA strand by PCR, followed by another chemical modification step where an electrochemical label attaches to the "handle". For this strategy, the main requirements are that the selected chemical reaction is orthogonal to other reactive groups present in the DNA molecule, is compatible with aqueous solutions, and is quantitative. "Click" chemistry meets all of the above requirements and has become a universal tool for the modification of DNA and proteins. Click chemistry is a reaction between an azide and an alkyne that usually yields a covalent product - 1,5-disubstituted 1,2,3-triazole catalyzed by copper(I). For copper-free "click" reactions, a sterically strained alkyne can be reacted with an azide, or trans-cyclooctene can be coupled with tetrazine ("third-generation click chemistry"). Any of the alkyne, trans-cyclooctene, azide, or tetrazine handle can be introduced into the DNA by a PCR step in which the corresponding modified nucleotide (see Compounds 1 - 8) is introduced, followed by a click reaction with an electrochemical label containing the other corresponding reactive group. The reactive group can be linked to the redox label via a carbon chain or an ethylene oxide (PEG) chain (Compounds 9 - 12).

Chem.

Chem.

Chem.

Chemical formula

[0088] In an alternative embodiment, the nucleotide itself is the reporter by monitoring changes in the tunneling current between biased electrodes. The chemistry of the nucleotide entering the polymerase and the change in the enzyme structure when the nucleotide enters the binding pocket cause a chemical shift in the tunneling efficiency that results in a change in the tunneling current used to identify the base present. Alternative modifications of DNA can be used to enhance the change in tunneling efficiency, for example, using a polymer backbone (PNA) instead of a deoxyribose backbone (DNA), because the uncharged backbone results in a more significant change in the electric field for the standard bases. Other chemistries can also be used with the ultimate goal of maximizing the disruption to the tunneling current when the base enters the sensing zone.

[0089] In other embodiments, the front end of the electronic device can be arranged in a fully differential manner. When current is flowing through one electrode of the front end, a current having the same amplitude but different polarity is flowing through the second input electrode of the front end. This avoids the disturbance applied to both electrodes from being transmitted through the signal path. Second, high-pass characteristics can be integrated into the first stage of the front end. This avoids differential DC current (from current flowing through tunneling or polymerase) from overloading the signal path of the front end. In other words, it avoids the signal being pushed to the limit of the measurement range due to those "parasitic" DC currents. The change in current from the base that needs to be detected is transmitted through the high-pass and processed within the electronic signal chain.

[0090] The methods of various embodiments also include RNA sequencing. The RNA can be processed upstream with a reverse transcriptase (RT) enzyme to create cDNA, and then the cDNA can be read using an immobilized DNA polymerase. Alternatively, the RT enzyme can be immobilized on the surface, and when the RNA sequence is replicated, the incorporation of redox-modified dNTPs by the RT enzyme is used to determine the original RNA template.

[0091] In other embodiments, a method of sequencing polynucleic acids using an electrochemical nanoelectrode sensor involves an array of immobilized enzymes, the activity of which is adjusted via external environmental parameters to assist their synchronization. This results in a device capable of reading long reads with single-base pair resolution and high fidelity. This is achieved by relying on multiple enzymes within the sensing zone configured to capture and translocate the nucleic acid of interest across the sensor sensing zone such that all enzymes function either in parallel or in concert. This parallel processing generates a higher signal compared to the signal generated by translocating a single polynucleotide strand per sensor. The multiple enzymes are attached to the surface in the vicinity of the electrical sensor and act as controlled localization sites to carry the nucleic acid to the sensing zone while providing a controlled translocation rate within the sensing zone. External control parameters can be used to synchronize the functions of the multiple enzymes within the sensing zone for high fidelity.

[0092] FIG. 20 shows a method and system for nucleic acid sequencing 2000 via immobilized enzymes, showing current versus voltage plots 2060 for different identification NTPs 2070 and associated current amplitudes 2030 versus time. The system includes bases (2002, 2004, 2006) and polymerase 2010 (e.g., 22, 68 shown in other figures). The bases include a first electrode 2002 (e.g., electrode 20 shown in other figures), a second electrode 2004 (e.g., electrode 18 shown in other figures), and a dielectric or insulator 2006 (e.g., dielectric 16 shown in other figures) configured to create a sensing zone 2008. When polymerase 2010 is activated, polymerase 2010 binds to base strand 2012 (e.g., 78 shown in other figures) at the point where complementary strand 2014 (e.g., 76 shown in other figures) ends and base strand 2012 begins. When bound, the electrodes 2002, 2004 can sense current such that the relationship between current 2032 and time 2034 can be viewed in graph 2030. This is due to the identification label (dNTP) 2070 used for each labeled nucleotide (2062, 2064, 2066, 2068) each having a different current-voltage relationship (2072, 2074, 2076, 2078). The electroactive labels (2062, 2064, 2066, 2068) have distinguishable electrochemical properties.

[0093] The shuttle detection mechanism includes two electrodes 2002, 2004 separated by a dielectric 2006 of nanoscale thickness. The electrodes are held at different voltages to enable electron transfer via the labels. The small space between the two electrodes is called the sensing zone 2010, which is small enough for electroactive molecules (2062, 2064, 2066, 2068) to interact with both electrodes 2002, 2004 and complete an electrical circuit. When multiple polymerases 2010 are immobilized near the dielectric 2006, they attract the base strands 2012 and align them at the ends of the complementary pairs 2014. While electroactive molecules (2062, 2064, 2066, 2068) are present within the sensing zone, electrons can "shuttle" between the two electrodes 2002, 2004, generating an increased current signal from multiple electroactive molecules (2062, 2064, 2066, 2068) that is much higher than the signal expected from a single electron transfer event. This mechanism can be considered a limited case of redox cycle amplification where electroactive molecules diffuse back and forth between the electrodes to generate an amplified electrical signal. When nucleotides are labeled with electroactive molecules, this sensing mechanism can be used to infer the sequence of NA.

[0094] Figure 21 shows an alternative method and system for nucleic acid sequencing in which a redox label attached to a base remains on the strand.

[0095] Figure 22 shows an alternative method and system for nucleic acid sequencing in which a redox label attached to the 3'-OH is cleaved after each base incorporation.

[0096] Figure 23 shows a method and system for parallel nucleic acid sequencing via an immobilized enzyme that shows current versus voltage plots for different discriminative NTPs and the associated current amplitudes versus time. By processing multiple NA strands in parallel, the flow of current is increased and can be measured with higher accuracy.

[0097] FIG. 24 shows a method and system for homogenous parallel nucleic acid sequencing via immobilized enzymes, showing the relevant current amplitude versus time. The term "homogenous" in one or more embodiments is used to describe the case where substantially all base strands and complementary strands are aligned such that the current increases due to the parallelism of electron transport across multiple labels.

[0098] FIG. 25 shows a method and system for phase-shifted parallel nucleic acid sequencing via immobilized enzymes, showing the relevant current amplitude versus time. The term "phase-shifted" in one or more embodiments is used to describe the case where substantially all fewer base strands and complementary strands are aligned such that the current decreases due to different electron transport characteristics across multiple different labels that are not constructively added.

[0099] FIG. 26 shows a current-versus-voltage plot and associated exemplary distinguishable electroactive labels. Several electroactive labels as well as labeled nucleotides and nucleosides were synthesized and their electrochemical properties were tested by recording cyclic voltammograms at a Pt electrode in aqueous solution.

[0100] A distinct signal generated by ferrocene-labeled dUTP in aqueous solution was recorded with a nanogap sensor. Electrode 1 was held at 0.45 V, while electrode 2 was held at 0.05 V. The experiment was performed with a Sutter patch clamp instrument. The higher current signal corresponds to ferrocene molecules within the sensing zone, while a lower constant current is generated by ferrocene molecules moving randomly within the bulk solution.

[0101] FIG. 27 shows the current versus time for electrode 1, the current versus time for electrode 2, and the differential current versus time 2700 for both electrode 1 and electrode 2. Spike 2702 is associated with an electron transport molecule (e.g., a ferrocene molecule) within the sensing zone, while the lower "noise" 2704 is associated with electron transport molecules (e.g., ferrocene molecules) within the bulk solution.

[0102] In one embodiment, an enzyme such as a biological polymerase is used to lower the target polynucleic acid to the sensing zone and control its translocation rate during sequencing. However, this embodiment can sequence only one polynucleic acid strand at a time per sensor and thus relies on the single-molecule sensitivity of the sensor. A way to increase the electrical signal created by electroactive labels and enable a larger sensing zone that is easier to manufacture is to enable simultaneous sequencing of multiple copies of the polynucleic acid. However, multiple enzymes may not start processing the polynucleic acids accurately simultaneously and may become further "out of sync" as sequencing progresses, leading to sequencing errors. This problem can be solved by using external parameters to control the activity of the enzyme. Such external parameters include, for example, temperature, light, small molecules, and inhibitors including synthetic or biological polymers (aptamers, peptides, proteins, cofactors), pH, and ion gradients including metal ions. These external parameters can be applied alone or in combination of two or more parameters to control the enzyme. In one or more embodiments, these control parameters can induce rapid, reversible, and iterable changes in enzyme function.

[0103] Multiple methods for modulating enzyme activity have been reported in the literature and include (a) site-specific conjugation of small molecules or polymers, (b) changing the pH in the vicinity of the enzyme electrochemically or using light and photoacids, (c) photoinduced conformational changes, (d) use of photoswitchable inhibitors, (e) metal-ion-induced conformational changes of aptamer inhibitors, (f) reversible binding of inhibitors, such as PMT technology by Nanohelix, or aptamers used by New England Biolabs and Soma Logic in hot-start polymerase chain reactions, and (g) changing the temperature of the reaction solution to decelerate or accelerate enzyme function.

[0104] In one or more embodiments, one or more of the above methods can be used to repeatedly and reversibly switch enzymes "on" and "off" to synchronize their activities and minimize their phase shifts.

[0105] In one embodiment, the immobilized enzyme (a) captures the strand of the target NA, (b) lowers it to the vicinity of the electrical sensor, and (c) translocates the strand across the sensor at a constant rate in units of one at a time. Several classes of enzymes, including polymerase, exonuclease, endonuclease, deoxyribonuclease, ribonuclease, helicase, and CRISPS-Cas type and related proteins, can meet this requirement.

[0106] Figure 28 shows a plurality of primers shifted to provide overlap between sequenced fragments.

[0107] To match the lengths of the fragments that can be reliably sequenced before the phase of the process shifts, a plurality of different primers can be designed to be shifted so that there is always an overlap between the sequenced fragments. In this situation, the DNA template sample is divided into different groups, each of which obtains its own sample preparation procedure such that the length of the double-stranded segment is a known interval shorter than the reliable read length (e.g., the length of in-phase enzyme activity). Then, each group of the DNA samples is added to a different set of sensors, and these sensors read the sequence starting from the ends of the double-stranded segments on the template. As a result, the most reliable reads (when the enzymes are in phase) between different groups are shifted along the length of the template. By comparing the sequences between different groups, the full-length sequence of the target DNA can be aligned and assembled.

[0108] Figure 29 shows the current versus time for Group 1, Group 2, and the full-length reads from Figure 28.

[0109] The following is an explanation of exemplary embodiments.

[0110] In the combination of enzyme, native NA, and labeled nucleotides, multiple copies of polymerase are immobilized on the surface of the electrical sensor. The unlabeled strand of nucleic acid is captured by the enzyme and localized on the sensor by the enzyme. The reaction components necessary for NA extension, including labeled nucleotides and primers, are added. When the labeled nucleotides are incorporated into the complementary NA strand, they are temporarily "paused" in the sensing zone of the sensor, resulting in a change in current. Labeled nucleotides floating in the solution contribute to the background current but do not generate a distinct signal due to their random Brownian motion. During the extension process, an external stimulus is applied to repeatedly switch all the enzymes on and off. Each switching event resets the enzymes so that they work in concert.

[0111] The label can be attached to the nucleotide via a linker at any of the base, triphosphate, or sugar ring. When attached to the base or 2'-OH, the label remains on the strand after incorporation. When attached to the triphosphate or 3'-OH, the label is cleaved by the polymerase during incorporation, allowing strand extension. This is advantageous as only one label is immobilized near the sensor, generating a distinct signal. The label is designed to be specific for different types of nucleotides.

[0112] In certain sequencing techniques involving modified nucleotides, there are often limitations on the length of the fragment that can be sequenced due to the distortion of the NA structure caused by additional functional groups on the nucleotide. As these distortions accumulate, the extension of the NA stops. In one or more embodiments, a multi-enzyme approach is used that results in a higher electrical signal generated by a collection of electroactive molecules, thereby enabling the use of a mixture of labeled and native nucleotides. For example, it is possible to have approximately 80% of the dNTPs unlabeled and only approximately 20% labeled. The labeled and unlabeled nucleotides are randomly incorporated into the copies of the growing DNA strand, thus causing less disruption to the natural structure of the NA and allowing for longer reads. At the same time, a signal sufficient to enable nucleotide assignment is created.

[0113] In the combination of enzyme, labeled NA, and native nucleotides, multiple copies of the polymerase are immobilized on the surface of the electrical sensor. The strand of nucleic acid labeled with an electroactive label is captured by the enzyme and localized on the sensor by the enzyme. Reaction components for NA extension, including native nucleotides and primers, are added. When nucleotides complementary to those in the labeled strand are incorporated, the label is translocated across the sensor by polymerase activity, temporarily "pauses" in the sensing zone of the sensor, resulting in a current change. Native nucleotides floating in the solution do not contribute to the background current and thus increase the signal-to-noise ratio compared to the first embodiment. An external stimulus is applied during the extension process to repeatedly switch all the enzymes on and off. Each switching event resets the enzymes and thus ensures that they work in concert. The label can be attached to the nucleotide via a linker to the sugar ring via a base or the 2'-OH position.

[0114] In the combination of exonuclease and labeled NA, multiple copies of the exonuclease are immobilized on the surface of the electrical sensor. The strand of nucleic acid labeled with an electroactive label is captured by the enzyme and localized on the sensor by the enzyme. Reaction components necessary for NA digestion are added. The labeled NA strand moves across the sensor by the action of the enzyme, the labeled nucleotides are cleaved and diffuse. The signal occurs when the label is temporarily "paused" primarily within the active pocket of the enzyme within the sensing zone of the sensor before the label is cleaved.

[0115] Alternatively, the enzyme may be immobilized further away from the sensing zone, and the signal is generated by the diffusing of the cleaved labeled nucleotides into the sensing zone. The higher surface area available for immobilization of the enzyme and NA results in a stronger signal generated by more labeled molecules.

[0116] Figure 30 shows one embodiment of a structure configured to sense an NA sequence. In this exemplary shape of such a sensor, the enzyme is immobilized (e.g., via silane chemistry) on the oxide surface of a well structure that includes two electrodes separated by a thin dielectric layer (aluminum oxide). Labeled nucleotides cleaved by the action of the exonuclease diffuse around the well, and some of them enter the sensing zone before diffusing. Here, the rate of nucleotide cleavage by the enzyme must be slower than diffusion in order to achieve a distinct signal from each cleaved nucleotide. By controlling external environmental parameters (including temperature, light, inhibitors including small molecules and synthetic or biological polymers (aptamers, peptides, proteins, cofactors), pH, and ionic gradients including metal ions), the rate of the enzyme can be slowed down to achieve optimal signal resolution.

[0117] Another exemplary flow includes (1) adding all nucleotides to the solution, (2) adding multiple primers to the solution, (3) activating the enzyme, and (4) measuring electrical properties related to the activation time to determine the sequence based on the electrochemical properties of electroactive molecules.

[0118] In this application, electroactive molecules include redox molecules, and redox signals include electrical signals such as changes in current. Poly nucleic acids (NA) include DNA, and nucleotides include dNTPs.

[0119] The processes, methods or algorithms disclosed herein can be realized / implemented by a processing device, a control device, or a computer that can include any existing programmable electronic control unit or a dedicated electronic control unit. Similarly, the processes, methods or algorithms can be stored as executable data and instructions by a control device or a computer in many forms including, but not limited to, information permanently stored in a non-writable storage medium such as a ROM device, and information changeably stored in a writable storage medium such as a floppy disk, a magnetic tape, a CD, a RAM device, and other magnetic and optical media. The processes, methods or algorithms can also be implemented as executable software objects. Alternatively, the processes, methods or algorithms can be wholly or partially embodied using suitable hardware components such as application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), state machines, control devices, or other hardware components or devices, or combinations of hardware, software and firmware components.

[0120] While the exemplary embodiments have been described above, it is not intended that these embodiments describe all possible forms encompassed by the claims. The language used herein is a language of description rather than of limitation, and it is understood that various changes may be made without departing from the spirit and scope of the disclosure. As previously mentioned, the features of the various embodiments may be combined to form further embodiments of the disclosure that may not be explicitly described or illustrated. Although the various embodiments may be described as providing advantages over other embodiments or prior art implementations with respect to one or more desired characteristics, those skilled in the art will recognize that one or more features or characteristics may be compromised in order to achieve the desired overall system characteristics that depend on a particular application and implementation. These characteristics can include, but are not limited to, cost, strength, durability, life cycle cost, marketability, appearance, packaging, size, serviceability, weight, manufacturability, ease of assembly, etc. Accordingly, as long as any embodiment is not described as less desirable than other embodiments or prior art implementations with respect to one or more characteristics, these embodiments are not outside the scope of the disclosure and may be desirable for a particular application.

Claims

**Claim 1** A method for nucleic acid sequencing, comprising: a first electrode, a second electrode, a dielectric member positioned between the first electrode and the second electrode and having a size such that electroactive molecules can interact with both the first electrode and the second electrode to complete an electrical circuit, creating a sensing zone, and two or more proteins immobilized on the surface of the dielectric member, each of the two or more proteins capturing a polynucleotide chain, transporting the polynucleotide chain into the sensing zone, and translocating the polynucleotide chain across the sensing zone at a constant rate, one nucleotide at a time providing at least one device comprising; applying a current to the first electrode and the second electrode, wherein the first electrode is held at a first voltage and the second electrode is held at a second voltage, thereby enabling electron transfer via an electroactive label covalently bound to a nucleotide; exposing the two or more proteins to a sample containing the polynucleotide chain; and detecting the current versus time of the first electrode and the second electrode to determine when the nucleotide having the electroactive label is within the sensing zone A method comprising the steps of: **Claim 2** The method of claim 1, further comprising applying at least one external parameter to the at least one device to reversibly and / or repeatedly regulate the activity of the two or more proteins. **Claim 3** The method of claim 2, wherein the applying step reversibly and / or repeatedly induces the two or more proteins to transition from an active state to an inactive state to synchronize the activity of the two or more proteins and maintain the two or more proteins in phase with each other. **Claim 4** The method of claim 3, wherein the at least one external parameter is selected from the group consisting of site-specific conjugation of small molecules or polymers, changes in pH in the vicinity of the protein, photoinduced conformational changes, use of photoswitchable inhibitors, metal ion-induced conformational changes of aptamer inhibitors, reversible binding of inhibitors, and changes in temperature of the sample containing the polynucleotide chain. **Claim 5** The method according to claim 4, wherein the polynucleotide chain translocated by the two or more proteins is aligned such that current increases due to parallelism of electron transport across a plurality of labels.

6. The method according to claim 1, wherein the protein is selected from the group consisting of DNA polymerase, RNA polymerase, ribosome, single-stranded binding protein, topoisomerase, helicase, nuclease, and CRISPR protein.

7. The method according to claim 1, wherein the electroactive label is covalently bonded to a nucleotide present in the polynucleotide chain.

8. The method according to claim 1, wherein the electroactive label is covalently bonded to a free nucleotide added to the sample containing the polynucleotide chain, and the two or more proteins incorporate the free electroactive label nucleotide into the polynucleotide chain within the sensing zone.

9. The method according to claim 1, wherein one of up to four different electroactive labels, each having a different current-voltage relationship, is covalently bonded to each nucleotide having a specific nucleotide base such that nucleotides having adenine, thymine, cytosine, and guanine bases are electrochemically distinguishable from each other.

10. A system for nucleic acid sequencing, comprising: a first electrode; a second electrode; a dielectric member positioned between the first electrode and the second electrode and having a size such that electroactive molecules can interact with both the first electrode and the second electrode to complete an electrical circuit, creating a sensing zone; two or more proteins immobilized on the surface of the dielectric member, each of the two or more proteins capturing a polynucleotide chain, transporting the polynucleotide chain into the sensing zone, and translocating the polynucleotide chain across the sensing zone at a constant rate, one nucleotide at a time; at least one device comprising: applying a current to the first electrode and the second electrode, holding the first electrode at a first voltage, and holding the second electrode at a second voltage to enable electron transfer through electroactive labels covalently bonded to nucleotides. Exposing the two or more proteins to a sample containing the polynucleotide strand, wherein the two or more proteins capture the polynucleotide strand and transport it into the sensing zone, and translocate the strand across the sensing zone at a constant speed, one nucleotide at a time Detecting the current versus time of the first electrode and the second electrode to determine when the nucleotide having the electroactive label is within the sensing zone, and Applying at least one external parameter to the at least one device to reversibly and / or repeatedly regulate the activity of the two or more proteins A control device configured to perform A system comprising

11. The system according to claim 10, wherein the step of applying the function of the control device induces the two or more proteins to transition from an active state to an inactive state, synchronizes the activity of the two or more proteins, and maintains the proteins in phase with each other

12. The system according to claim 11, wherein the polynucleotide strands are aligned in phase with each other to form aligned polynucleotide strands such that the current increases due to the parallelism of electron transport across a plurality of labels

13. The system according to claim 10, wherein the control device is further configured to apply the at least one external parameter to reversibly maintain the proteins out of phase with each other

14. The system according to claim 13, wherein the polynucleotide strands are out of phase and aligned such that the current decreases due to different electron transport characteristics across a plurality of different labels that are not constructively added

15. The system according to claim 10, wherein the at least one external parameter is selected from the group consisting of site-specific conjugation of small molecules or polymers, change in pH in the vicinity of the protein, photoinduced conformational change, use of a photoswitchable inhibitor, metal ion-induced conformational change of an aptamer inhibitor, reversible binding of an inhibitor, and change in temperature of the sample containing the polynucleotide strand

16. The system according to claim 10, wherein the electroactive label is covalently bonded to a nucleotide present in the polynucleotide strand

17. The system of claim 10, wherein the electroactive label covalently binds to free nucleotides added to the sample containing the polynucleotide chain, and the two or more proteins incorporate the free electroactive label nucleotides into the polynucleotide chain within the sensing zone.

18. A method for forming a device for nucleic acid sequencing, comprising: providing at least one device including a first electrode, a second electrode, and a dielectric member positioned between the first electrode and the second electrode; configuring the dielectric member to operate as a sensing zone sized such that electroactive molecules can interact with both the first electrode and the second electrode to complete an electrical circuit; and immobilizing two or more proteins on the surface of the dielectric member, each of the two or more proteins capturing a polynucleotide chain, transporting the polynucleotide chain into the sensing zone, and translocating the chain across the sensing zone one nucleotide at a time at a constant rate comprising a method.

19. The method of claim 18, wherein the first electrode is held at a first voltage and the second electrode is held at a second voltage, thereby enabling electron transfer via an electroactive label, and the electroactive label is covalently bound to a nucleotide.

20. The method of claim 18, wherein the protein is selected from the group consisting of DNA polymerase, RNA polymerase, ribosome, single-stranded binding protein, topoisomerase, helicase, nuclease, and CRISPR protein.

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