Enzyme transposase in nanogap with 3'-ester

Abstract

Methods for nucleic acid sequencing include providing at least one device comprising a first electrode and a second electrode separated by a dielectric layer, and a polymerase attached to a surface of the dielectric layer. The dielectric layer induces an electroactive molecule to interact with the electrodes to complete an electrical circuit. The polymerase targets a polynucleotide strand to the dielectric layer. A sample comprising the polynucleotide strand and a modified nucleotide having an electroactive label covalently bound to a 3'-OH of a sugar ring of the nucleotide via an ester group is provided to the at least one device. An electrical potential is applied to each electrode to induce a flow of electrons between the electrodes to produce a measurable electrical signal when the electroactive label is present in the dielectric layer. The electrical signal from the electrodes is detected to determine when the modified nucleotide is present in the dielectric layer.

Description

Invention Field

[0001] In at least one aspect, this disclosure relates to systems, apparatus, and methods for nucleic acid sequencing. Background of the Invention Single-base-pair resolution DNA sequencing is a critical goal in biotechnology. To date, most techniques require significant reconstruction or repeated runs of sequences from small reads to achieve fidelity. A need arises for a technique that combines the scalability and speed of semiconductor-based electrical detection with the high accuracy of SbS technology to provide long reads at single-base-pair resolution. Invention Overview Methods for nucleic acid sequencing are provided in various aspects. The method includes providing at least one device comprising a first electrode and a second electrode separated by a dielectric layer. The dielectric layer defines a sensing region between the first and second electrodes. A polymerase is attached to the surface of the dielectric layer. The polymerase targets a polynucleotide chain to the dielectric layer. The method further includes providing the at least one device with a sample comprising a polynucleotide chain and at least one modified nucleotide having an electroactive label covalently bound to the 3'-OH group of the sugar ring of the nucleotide via an ester group; applying a first potential to the first electrode and a second potential to the second electrode to induce an electron flow between the first and second electrodes to generate a measurable electrical signal when the electroactive label is present in the sensing region; and detecting the electrical signal from the first and second electrodes to determine when the modified nucleotide is present in the sensing region.

[0004] In another aspect, a system for nucleic acid sequencing is provided. The system includes at least one device comprising: at least one electrochemical nanoelectrode sensor including a first electrode, a second electrode, and a dielectric layer defining a sensing region between the first and second electrodes. A polymerase is attached to the surface of the dielectric layer, and the polymerase targets a polynucleotide chain to the sensing region. The system further includes a controller configured to: direct a first current through the first electrode and direct a second current through the second electrode to induce an electron flow between the first and second electrodes to generate a measurable electrical signal when an electroactive label is present in the sensing region; provide the at least one device with a sample comprising a polynucleotide chain and at least one modified nucleotide having an electroactive label covalently bound to the 3'-OH group of the nucleotide's sugar ring via an ester group; and detect the first current flowing from the first electrode and the second current flowing from the second electrode.

[0005] In other aspects, a method for forming a system for nucleic acid sequencing is provided. The method includes the steps of: providing a first electrode and a second electrode; placing a dielectric layer between the first and second electrodes; attaching a polymerase to the surface of the dielectric layer, wherein the polymerase incorporates a modified nucleotide into a polynucleotide chain, the modified nucleotide having an electroactive label covalently bound to the 3'-OH group of the nucleotide's sugar ring via an ester group; and configuring the electrodes to detect changes in current when the electroactive label is present within a sensing region. Brief description of the attached diagram Figure 1 The concept of using electrochemical sensors, immobilized polymerases, and labeled nucleotide sequencing is illustrated.

[0007] Figure 2 Schematic diagrams of three different geometries used to modify planar electrode pairs are shown.

[0008] Figure 3 Schematic diagrams of two different geometries for a non-planar design are shown, wherein the electrode pairs are fabricated as a stack in the form of holes (A), and wherein the holes are filled on one side (B).

[0009] Figure 4 A method for synthesizing 3'-esterified dNTPs with an electroactive label (E) is shown.

[0010] Figure 5 Examples of electroactive markers (E) and connectors (L) are shown.

[0011] Figure 6 The first step of a method for synthesizing a ferrocene-labeled reversible terminator (A3) with an ester group is shown.

[0012] Figure 7 It shows Figure 6 The second step of the method (synthesizing compound A2 from A1).

[0013] Figure 8 It shows Figure 6 The third step of the method (synthesizing compound A3 from A2).

[0014] Figure 9 The first step of a method for synthesizing a ferrocene-labeled reversible terminator (B5) with an ester group is shown.

[0015] Figure 10 It shows Figure 9 The second step of the method (synthesizing compound B2 from B1).

[0016] Figure 11 It shows Figure 9 The third step of the method (synthesizing compound B3 from B2).

[0017] Figure 12 It shows Figure 9 The fourth step of the method (synthesizing compound B4 from B3).

[0018] Figure 13 It shows Figure 9 The fifth step of the method (synthesizing compound B5 from B4).

[0019] Figure 14 A method for synthesizing a ferrocene-labeled reversible terminator (C3) with an ester group and no linker is shown.

[0020] Figure 15 Cyclic voltammograms (CVs) for electroactive labels are shown: anthraquinone carboxylic acid (green), methylene blue (blue), ferrocene carboxylic acid (orange), and phenothiazine carboxylic acid (red). CVs were recorded in a 10 mM phosphate buffer solution containing 100 mM KCl and 10% DMSO.

[0021] Figure 16 An embodiment of a readout electronics for an electrode display is shown. Invention Details As requested, detailed embodiments of the invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely examples of the invention and may be embodied in various and alternative forms. The drawings are not necessarily drawn to scale; some features may be enlarged or reduced to show details of specific components. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but rather serve only as a representative basis for teaching those skilled in the art to employ the invention in various ways.

[0023] Except as expressly indicated in the embodiments or elsewhere, all quantities indicating the amount of reaction and / or materials or conditions used in this specification shall be understood to be modified by the word “about”. The initial definition of an abbreviation or other acronym applies to all subsequent uses of the same abbreviation herein, and with necessary modifications, applies to the conventional grammatical variations of the originally defined abbreviation; and, unless expressly stated otherwise, the measurement of a property is determined by the same technique used previously or subsequently for the same property.

[0024] Unless otherwise stated, 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 pertains.

[0025] It should also be understood that this disclosure is not limited to the specific embodiments and methods described below, as specific components and / or conditions may, of course, change. Furthermore, the terminology used herein is for describing particular embodiments only and is not intended to be limiting in any way.

[0026] It must also be noted that, as used in the specification and appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. For example, references to components in the singular are intended to include multiple components.

[0027] The terms “or” and “and” can be used interchangeably and can be understood as meaning “and / or”.

[0028] The term "comprising" is synonymous with "including," "having," "containing," or "characterized in." These terms are inclusive and open-ended and do not exclude additional, unlisted elements or method steps.

[0029] The phrase "consisting of" excludes any element, step, or component not specified in the claim. When this phrase appears in the body clause of a claim, rather than immediately following the preamble, it only limits the element described in that clause; other elements are not excluded from the claim as a whole.

[0030] The phrase “consistent essentially of” limits the scope of the claim to the specified materials or steps, plus those materials or steps that do not substantially affect the essential and novel characteristics of the subject matter for which protection is sought.

[0031] The terms “comprising,” “consisting of,” and “substantially composed of” may be used interchangeably. When one of these three terms is used, the subject matter disclosed herein and for which protection is sought may include the use of any of the other two terms.

[0032] 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, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can 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 naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiments. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be conferred before or after polymer assembly. The sequence of the nucleotide may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeled components.

[0033] The term "sequence identity" or "identity" refers to the specified percentage of identical residues in two nucleic acid or amino acid sequences when compared for maximum correspondence within a specified comparison window, as measured by sequence alignment algorithms or visual inspection. When sequences differ in terms of conserved substitutions, the percentage sequence identity can be adjusted upwards to correct for the conservatism of the substitutions. Sequences that differ by such conserved substitutions are said to have "sequence similarity" or "similarity." The means of preparing such adjustments are well known to those skilled in the art. Typically, this involves scoring the conserved substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity.

[0034] The term "comparison window" refers to a segment with at least about 20 adjacent positions, whereby, after optimal alignment of two sequences, the sequence can be compared with a reference sequence having the same number of adjacent positions. In an improved version, the comparison window is 15 to 30 adjacent positions, whereby, after optimal alignment of two sequences, the sequence can be compared with a reference sequence having the same number of adjacent positions. In another improved version, the comparison window is typically about 50 to about 200 adjacent positions, whereby, after optimal alignment of two sequences, the sequence can be compared with a reference sequence having the same number of adjacent positions.

[0035] The term “complementarity” or “complement” refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence via conventional Watson-Crick or other non-traditional types. Percentage 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, 5, or 6 residues are 66.67%, 83.33%, and 100% complementary, respectively). “Completely complementary” means that all adjacent residues in the nucleic acid sequence are hydrogen-bonded to the same number of adjacent residues in the second nucleic acid sequence. As used herein, “substantially complementary” refers to a degree of complementarity of at least 40%, 50%, 60%, 62.5%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% in regions of 4, 5, 6, 7, and 8 nucleotides, or a percentage between the two, or to two nucleic acids hybridizing under stringent conditions.

[0036] As used herein, the terms “transposin,” “transporter,” “enzyme,” and “protein” refer to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein capable of translocating a polynucleotide chain. Examples of proteins capable of translocating polynucleotide chains include DNA polymerases, RNA polymerases, ribosomes, single-strand binding proteins, topoisomerases, helicases, nucleases, exonucleases, endonucleases, zinc finger nucleases, RNA-guided DNA endonucleases, transcription activator-like effector nucleases, CRISPR proteins, and combinations thereof.

[0037] Unless otherwise explicitly stated: all R groups (e.g., R...) i (where i is an integer) all include 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", where R', R" and R"' are C 1-10 Alkyl or C 6-18 Aryl group; single letter (e.g., "n" or "o") is 1, 2, 3, 4, or 5; in the compounds disclosed herein, the CH bond may be substituted by: 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", where R', R" and R"' are C 1-10 Alkyl or C 6-18Aryl groups; indications of a positively charged portion or structure imply the presence of one or more negative counterions to balance the charge, and similarly, indications of a negatively charged portion or structure imply the presence of one or more positive counterions to balance the charge; percentages, “parts” and ratios are by weight; the term “polymer” includes “oligomer,” “copolymer,” “terpolymer,” etc.; unless otherwise stated, the molecular weight provided for any polymer refers to the weight-average molecular weight; a description of a group or class of materials as suitable or preferred for a given purpose in conjunction with the present invention implies that a mixture of any two or more members of that group or class is equally suitable or preferred; a description of chemical composition refers to the composition at the time of addition to any combination specified in the description and does not necessarily exclude the chemical interactions of the components of the mixture after mixing; the initial definition of abbreviations or other abbreviations applies to all subsequent uses of the same abbreviation herein and, with necessary modifications, to the conventional grammatical variations of the abbreviations in their initial definition; and, unless otherwise expressly stated, the measurement of properties is performed by the same technique as previously or subsequently mentioned for the same property.

[0038] As used herein, the term "alkyl" refers to C 1-20 Linear, branched, cyclic, saturated, or at least partially and in some cases completely unsaturated (i.e., alkenyl and ynyl) hydrocarbon chains, including, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, vinyl, propynyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and propadienyl groups. "Lower alkyl" refers to an alkyl group having 1 to 8 carbon atoms, for example 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms (i.e., C16, C26, C36, C46, ​​C56, C66, C76, C86, C9 ... 1-8 Alkyl). Lower alkyl can also refer to any range between two carbon numbers listed above. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Higher alkyl can also refer to any range between two carbon numbers listed above.

[0039] As used herein, the term "aryl" refers to an aromatic substituent, which can be a single aromatic ring, or multiple aromatic rings fused together, covalently linked, or connected to a common linking group (e.g., but not limited to the methylene or ethylidene moiety). The common linking group can also be a carbonyl group (as in benzophenone) or an oxygen group (as in diphenyl ether). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, and diphenyl ether. Aromatic groups include heteroaryl groups, wherein one or more aromatic rings include heteroatoms (e.g., N, O, S, or Se). Exemplary heteroaryl groups include, but are not limited to, furanyl, pyridinyl, pyrimidinyl, imidazole, benzimidazolyl, benzofuranyl, benzothiophenyl, quinolinyl, isoquinolinyl, thiophenyl, etc. The aryl group may optionally be substituted with one or more aryl substituents (“substituted aryl”), which may be the same or different, wherein “aryl substituent” includes alkyl (saturated or unsaturated), substituted alkyl (e.g., haloalkyl and perhaloalkyl, such as but not limited to –CF3), cycloalkyl, aryl, substituted aryl, aralkyl, halogen, nitro, hydroxyl, acyl, carboxyl, alkoxy (e.g., methoxy), aryloxy, arylalkoxy, thioalkyl, thioaryl, thioaralkyl, amino (e.g., aminoalkyl, aminodialkyl, aminoaryl, etc.), sulfonyl and sulfinyl.

[0040] The terms “redox molecule,” “redox label,” “electroactive molecule,” and “electroactive label” are used interchangeably to refer to molecules that are redox species capable of undergoing reversible redox reactions under an applied potential.

[0041] It should also be understood that integer ranges explicitly include all intermediate integers. For example, the integer range 1-10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 includes 1, 2, 3, 4…97, 98, 99, 100. Similarly, when any range is invoked, the intermediate number of the increment between the upper and lower limits divided by 10 can be considered an alternative upper or lower limit. For example, if the range is 1.1 to 2.1, the numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be chosen as either the lower or upper limit. In the specific examples illustrated herein, concentrations, temperatures, and reaction conditions (e.g., pressure, pH, etc.) can be practiced with positive or negative 50% of the indicated values, rounded to three significant figures. In one improvement, concentration, temperature, and reaction conditions (e.g., pressure, pH, etc.) can be practiced with a value plus or minus 30% of the indicated value, rounded to three significant figures as provided in the examples. In another improvement, concentration, temperature, and reaction conditions (e.g., pH, etc.) can be practiced with a value plus or minus 10% of the indicated value, rounded to three significant figures as provided in the examples.

[0042] In the examples described herein, concentration, temperature, and reaction conditions (e.g., pressure, pH, flow rate, etc.) can be practiced with plus or minus 50% of the indicated values, rounded or truncated to two significant figures. In an improvement, concentration, temperature, and reaction conditions (e.g., pressure, pH, flow rate, etc.) can be practiced with plus or minus 30% of the indicated values, rounded or truncated to two significant figures. In yet another improvement, concentration, temperature, and reaction conditions (e.g., pressure, pH, flow rate, etc.) can be practiced with plus or minus 10% of the indicated values, rounded or truncated to two significant figures.

[0043] In this application, electroactive molecules include redox molecules, and redox signals include electrical signals such as changes in current. Polynucleic acids (NAs) include DNA, and nucleotides include dNTPs.

[0044] The processes, methods, or algorithms disclosed herein may be deliverable to / implemented by a processing device, controller, or computer, which may include any existing programmable electronic control unit or dedicated electronic control unit. Similarly, processes, methods, or algorithms may be stored as data and instructions executable by a controller or computer in various forms, including but not limited to information permanently stored on non-writable storage media such as ROM devices, and information variablely stored on writable storage media such as floppy disks, magnetic tapes, CDs, RAM devices, and other magnetic and optical media. Processes, methods, or algorithms may also be implemented in an executable software object. Alternatively, processes, methods, or algorithms may be embodied, in whole or in part, using suitable hardware components such as application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), state machines, controllers, or other hardware components or devices, or a combination of hardware, software, and firmware components.

[0045] Throughout this application, when references are made to publications, the disclosures of those publications, in their entirety, are incorporated herein by reference to provide a more comprehensive description of the prior art in the field to which this invention pertains.

[0046] Single-base resolution DNA sequencing is a crucial goal in biotechnology. To date, most techniques require reconstruction of sequences from small reads or numerous repetitive runs to achieve fidelity. Sequencing-by-synthesis (SbS) is currently the gold standard among DNA sequencing methods due to its high accuracy. It employs the incorporation of nucleotides that are fluorescently labeled at bases and protected at the 3'-OH position. These modified nucleotides are called reversible terminators. The DNA sequence is determined by adding one modified nucleotide per synthesis cycle, through the synthesis of a complementary DNA strand alongside the template strand. Each cycle consists of: incorporation of the modified nucleotide into the growing DNA strand, optical imaging to identify the type of nucleotide, and removal of the label and protecting group, thus making the 3'-OH group available for the addition of the next nucleotide.

[0047] The main limitation of the SbS method is its long cycle time, primarily due to the need for optical imaging of large areas of the flow cell. The accumulation of "scars" (the portions of the linker that attach the label to the bases and remain on the growing DNA strand after label removal) leads to the eventual termination of complementary strand synthesis, thus limiting the size of the sequencing fragment to ~150 bases. Other established sequencing methods offer longer read lengths and faster sequencing times.

[0048] Single-molecule real-time (SMRT) sequencing technologies, such as those developed by PacBio, use enzymes immobilized on the surface of a zero-mode waveguide (ZMW). When the sequencing reaction begins, the enzyme incorporates nucleotides modified with fluorophores (which are attached to triphosphate groups) into the growing DNA strand. While the labeled nucleotides briefly "pause" in the polymerase's active pocket, they generate a stronger fluorescence signal compared to the background signal from freely floating modified nucleotides in solution. A high-resolution camera on the ZMW records the fluorescence of the incorporating successive nucleotides in a cinematic manner. After each nucleotide incorporation, the fluorophore, along with the pyrophosphate group, is released from the growing DNA strand via triphosphate hydrolysis, leaving no "trace," allowing the next nucleotide with the fluorophore to enter. Due to specialized library preparation techniques, the same circular DNA fragments can be read multiple times, thus overcoming the high error rates associated with real-time sequencing.

[0049] Semiconductor sequencing technology represents a cost-effective and rapid benchtop sequencing system. Technologies such as those developed by IonTorrent use arrays of semiconductor chips to detect nucleotide incorporation events by sensing small pH changes. This technology requires no special enzymes or modification of natural nucleotides.

[0050] Nanopore sequencing platforms, such as those developed by Oxford Nanopore Technologies, record changes in electrical current as nucleic acids pass through biological nanopores. Each nucleotide possesses unique current modulation characteristics and can be identified without any labeling. This approach provides rapid sequencing of long DNA fragments, but challenges remain in achieving single-base resolution and sufficiently high accuracy.

[0051] Therefore, there is a need for a technology that combines the scalability and speed of semiconductor-based electrical detection with the high accuracy of SbS technology to provide long readings at single-base-pair resolution.

[0052] This article presents a system and method for nucleic acid sequencing that addresses the aforementioned needs. The system and method include an electrochemical nanoelectrode sensor, a biopolymerase that brings a polynucleotide chain to the vicinity of the sensor, and a modified nucleotide carrying an electroactive label. The label can be covalently attached to the 3'-OH group of the nucleotide via a cleavable ester group. During the incorporation of the modified nucleotide into the growing nucleic acid chain, the electroactive label can "idle" inside the sensor, generating a strong signal until the esterase function of the polymerase cleaves the ester group, thus removing the label from the sensor. The advantage of using 3'-esterified nucleotides is that the 3'-ester group provides an electroactive label with a longer residence time at the sensor through the relatively slow cleavage by the polymerase, resulting in a stronger signal. The traceless removal of the label from the growing nucleic acid chain allows for longer readouts.

[0053] The systems and methods provided herein may include devices capable of reading long reads at single-base-pair resolution. This disclosure may also incorporate the addition of transport proteins, such as biopolymerases, as a method for bringing polynucleotide chains into a detection device described in U.S. Patent No. 11,131,646, issued September 28, 2021, which is incorporated herein by reference in its entirety. The advantage of this approach is that the transport protein can act as a controlled localization site to bring the polynucleotide chain into the sensing region and simultaneously provide a controlled transport rate within the sensing region, both parameters that are controllable for single-base-pair resolution sequencing. The device may be an electrochemical sensor. Hereinafter, the terms device or electrochemical sensor may be used interchangeably to refer to the disclosed device.

[0054] Electrochemical sensors can comprise a first electrode and a second electrode separated by a nanometer-thick dielectric layer. The first and second electrodes can be maintained at two different potentials to induce electron transfer in the presence of electroactive molecules. The dielectric layer provides a small space between the first and second electrodes. This space is small enough for electroactive molecules to interact directly or indirectly with the first and second electrodes to complete the circuit. The small space above the dielectric layer between the first and second electrodes is referred to hereinafter as the sensing region. When redox molecules reside in the sensing region, electrons flow between the reduction and oxidation electrodes, generating an amplified current signal that is much higher than the signal predicted by a single electron transfer event. This mechanism differs from diffusion-based electron transfer, where electroactive molecules diffuse between the electrodes to generate a measurable electrical signal. When redox molecules reside in the sensing region, the amplified current signal generated by the resulting electron flow can be measured and used to identify the presence of redox molecules in the sensing region.

[0055] The electrode size should be small enough to suppress background signals from free labeled nucleotides in solution. Preferred electrode sizes in contact with the solution can be between 50 nm × 10 nm and 5 μm × 1 μm.

[0056] The key parameters are the dielectric layer thickness and the overlap between the two working electrodes. The dielectric layer thickness should be small enough to allow electron transfer in the presence of the electroactive label, while providing sufficient insulation between the electrodes to avoid short circuits. The preferred dielectric layer thickness can be from about 1 nm to about 10 nm. For example, the thickness of the dielectric layer can be approximately 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 approximately 10 nm. The overlap between electrodes should be kept to a minimum to avoid short circuits through pinholes in the dielectric layer.

[0057] Non-limiting examples of suitable materials for electrodes may include platinum, palladium, and titanium nitride.

[0058] Non-limiting examples of suitable materials for dielectric layers may include hafnium silicate 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.

[0059] Non-limiting examples of methods that can be used to fabricate sensing structures are described in U.S. Patent No. 11,131,646, which is incorporated herein by reference in its entirety. For example, the fabrication of a sensing structure may include sputtering or evaporation of electrode material, and the use of chemical and / or physical dry etching or wet etching to form the desired electrode shape. A thin dielectric layer between the electrode pairs can be deposited using atomic layer deposition with a thickness of 1 nm to 10 nm. Openings can be etched using physical and chemical dry etching (including, but not limited to, ion beam etching, inductively coupled plasma reactive ion etching, or magnetically enhanced reactive ion etching) to expose nanoscale gaps. Arrays of these sensors can be fabricated using this method to increase throughput and base recall accuracy.

[0060] The system and method presented herein utilize biopolymerases to replicate DNA by incorporating novel nucleotides as a means of localizing electroactive labels to the sensing region of an electrochemical sensor. Previous methods employed γ-phosphate-modified nucleotides. In contrast, this system and method use 3'-O-modified nucleotides and ester functional groups, which results in a reduction of the kinetics of nucleotide incorporation, thus achieving single-base resolution.

[0061] In one embodiment, an enzyme (e.g., DNA polymerase) capable of transporting polynucleotide chains and incorporating free nucleotides into the polynucleotide chains can be immobilized on the surface of a dielectric layer between two electrodes. Two different potentials can be applied to each electrode. Although labeled nucleotides floating in solution generate a background signal, this signal can be removed by passivating the electrode surface after enzyme immobilization to reduce the active electrode region outside the spot where the polymerase is attached. Alternatively, the signal from freely floating labeled nucleotides can be removed by an electronic filter. Because freely floating molecules are diffused at a much faster rate than the typical sampling frequency of electronic readout, their activity exhibits a time-averaged effect (e.g., a constant DC baseline), which can be removed by filtering slow responses. In contrast, incorporated nucleotides can remain in the sensing region for a longer period of time and thus generate transient events detectable as incorporation signals by electronic readout. After the ester bonds are hydrolyzed by the enzyme, the label can be removed from the sensing region and the signal can fall back to the background diffusion level until the next labeled nucleotide enters the enzyme's active pocket. The sensor can record the signal as a change in a current signal. Based on the voltage applied to the electrode and the signal characteristics generated by the label, the type of nucleotide incorporated can be inferred.

[0062] Non-limiting examples of redox molecules that can be used as markers include organometallic complexes, such as ferrocene and its derivatives, osmium and ruthenium complexes, and conjugated organic molecules, such as tetrathiofulvalene, methylene blue, anthraquinone, phenothiazine, aminophenol, nitrophenol, erythrosine B, ATTO MB2, etc. The redox species must undergo a reversible redox reaction under the applied potential to allow for the redox detection principle.

[0063] The enzyme used must be able to incorporate a 3'-O modified dNTP and subsequently cleave the ester bond to release the free 3'-OH, allowing for the addition of the next nucleotide. Non-limiting examples of suitable enzymes include family A polymerases that incorporate (2-aminoethoxy)-3-propionyl-dNTPs and allow for sequential DNA synthesis, as well as certain family B polymerases. Family A polymerases include BF, Bsu, KF, Taq, Tfl, Tth, T3, T5, T7, and EcPol I, Pol γ, Pol θ, and Polν. 9°N DNA and Vent polymerases can be suitable family B polymerases.

[0064] In various implementations, enzymes with altered activities, including but not limited to increased esterase rates or base specificity, can be generated through directed evolution. The nucleobases of the amino acids present in the active site encoding the enzyme of interest can be randomized, while other nucleobases are not. Such partially randomized libraries can be screened in vitro using droplet-based microfluidic screening or via in vivo hypermutation. In this way, enzymes with altered activities of interest can be identified.

[0065] Nucleotide reversible terminators with different 3'-O-blocking chemical groups are described in the prior art and have been incorporated into commercial products for SbS. Examples of such nucleotide reversible terminators include allyl, azidomethyl, (2-aminoethoxy)-3-propionyl, tert-butyldithiomethyl, amino, or 2-nitrobenzyl groups. However, there are no known reports of electroactive molecules attached to the 3'-ester group via dNTPs and their successful incorporation via polymerases.

[0066] Some systems can attach the label to the 2'-OH group of the sugar ring. Other systems may require additional modification of the dNTP at the 3'-OH with a blocking group to enable a reversible dNTP terminator. This approach also requires a hydrolysis step to release the redox moiety before they can be detected. Finally, this approach requires an additional step to chemically cleave the blocking group and release the free 3'-OH for sequencing.

[0067] In a preferred embodiment, the electroactive molecule is attached to the 3'-OH group of the sugar ring (e.g., via covalent bonding). Attaching the electroactive molecule to the 3'-OH group of the sugar ring eliminates the need for further modification of the dNTP by attaching a blocking group to the 3'-OH group of the sugar ring, making a reversible dNTP terminator possible. Indeed, in the systems and methods described herein, polymerization does not need to terminate after each addition of a modified nucleotide. Continuous synthesis enabled by the systems and methods described herein allows for faster sequencing. Furthermore, the systems and methods described herein do not require a hydrolysis step that releases the redox moiety before it can be detected. Instead, the systems and methods described herein generate a signal from the redox label while the label is still incorporated into the enzyme-template-dNTP complex and paused within the sensing region.

[0068] Instead of the additional step of chemically cleaving the blocking group and releasing free 3'-OH to continue sequencing, the system and method described herein combine the release of redox labels and the deblocking of 3'-OH via the action of the enzyme itself, without any additional steps or chemical reagents, thus simplifying the process.

[0069] Instead of modifying only one type of dNTP (dATP, dGTP, dCTP, or dTTP / dUTP) per DNA extension cycle, the system and method described herein allow modification of all four types of dNTPs in a single sample, thus reducing the number of cycles required to sequence a DNA strand to one-quarter.

[0070] Figure 1 An embodiment of a device 100 for sequencing nucleic acids according to the system and method described herein is shown. The device includes a first electrode 101, a second electrode 102, and a dielectric layer 103 disposed between the first and second electrodes. The device may also be referred to as an electrochemical sensor, a nanogap sensor, or a nanoelectrosensor. The space between the first electrode 101 and the second electrode 102 defines a sensing region 104. A protein 105, capable of transporting polynucleotide chains and incorporating free nucleotides into the polynucleotide chains, is immobilized on the surface of the dielectric layer 103. A solution containing polynucleotide chains 106 and freely floating modified nucleotides 107 is added to the device 100, each nucleotide labeled with an electroactive molecule 108. The electroactive molecule 108 can be covalently bonded to the 3'-OH group of the sugar of the nucleotide. Different potentials are applied to each of the first electrode 101 and the second electrode 102. After a potential is applied to the first and second electrodes, the freely floating modified nucleotides 107 can generate a background signal 109. In contrast, the modified nucleotide 107 incorporated into the polynucleotide chain is retained in the sensing region for a longer period of time than freely floating modified nucleotides 107. The incorporated modified nucleotide 107 thus generates a transient event during incorporation, which can be detected electronically as an incorporation signal 110. The type of incorporated modified nucleotide 107 is inferred based on the voltage applied to the electrode and the signal characteristics generated by a specific label. The sequence of the polynucleotide chain 106 is thus inferred from the signal characteristics generated by the label on each nucleotide.

[0071] According to certain embodiments, the protein immobilized to the surface of the dielectric layer can be an enzyme that can incorporate a 3'-O-modified dNTP and subsequently cleave the ester bond to release free 3'-OH, allowing for the addition of the next nucleotide. In the following, the terms protein, enzyme, or polymerase may be used interchangeably to refer to the protein immobilized to the surface of dielectric layer 103.

[0072] Figure 2Various embodiments of the device are shown, wherein the device includes planar electrodes. The device may have an open configuration 111, wherein a first electrode 101, a dielectric layer 103, and a second electrode 102 are linearly aligned along a surface. A solution containing polynucleotide chains and modified nucleotides can then flow through the surface containing the electrode pairs and the dielectric layer, wherein polymerase 112 is attached to the dielectric layer 103. In another embodiment, a channel 113 may be formed, wherein the first electrode 101, the dielectric layer 103, and the second electrode 102 are linearly aligned along the wall of the channel. A solution containing polynucleotide chains 106 and nucleotides can then flow through the channel. In yet another embodiment, a pore 114 may be formed in the device. The first electrode 101, the dielectric layer 103, and the second electrode 102 may be linearly aligned along the bottom surface of the pore, as shown below. Figure 2 As depicted in [the text]. A solution containing polynucleotide chains 106 and nucleotides can then be added to the wells. Still refer to [the text]. Figure 2 E1 refers to the first electrode 101, E2 refers to the second electrode 102, and D1 refers to the dielectric layer 103.

[0073] Figure 3 Multiple embodiments of a device using a non-planar design for electrodes are shown. (Reference) Figure 2 E1 refers to the first electrode 101, E2 refers to the second electrode 102, and D1 refers to the dielectric layer 103. In one embodiment, a hole 115 may be formed in the device. In this embodiment, the first electrode 101, the dielectric layer 103, and the second electrode 102 are fabricated as a stack in the form of a hole. Two stacks may be fabricated, one inside each wall of the hole. In another embodiment, a stack may be fabricated on each wall inside the hole 116. Only one stack may be accessed by a liquid sample. The accessible stack may have polymerase attached to the surface of the dielectric layer.

[0074] In various embodiments, the modified nucleotide 107 labeled with electroactive molecule 108 has the following formula: in: X is It's a single button, a double button, and a triple button. The bases are adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). L (connector) is either absent or contains a hydrocarbon chain of 1 to 1000 atoms, and may contain heteroatoms such as O, N, and S. n = 1-1000, R1 is H or OH, and R2 is a redox marker.

[0075] Figure 4 Synthetic strategies for generating electroactively labeled 3'-esterified dNTPs are outlined. Route A begins with the reaction of a 5'-protected nucleoside with a carboxylic acid compound incorporating an electroactive molecule and a linker to form a 3'-esterified nucleoside. This nucleoside is then deprotected and converted to a triphosphate in the next step. Alternatively, 5'-O-dimethoxytriphenylmethyl-dN-3'-O-succinic acid can be used as a starting material, and an electroactive molecule with an amino group can be coupled to a protected nucleotide derivative, followed by deprotection and triphosphorylation (Route B). For Route C, a 3'-ester can be formed first, followed by triphosphorylation, and finally, an attached electroactive molecule can be formed via an amide.

[0076] Examples of modified nucleotides 107 labeled with electroactive molecule 108 include compounds having the following formula: ,or Figure 5 Examples of electroactive labels and linker groups that can be used to modify modified nucleotides 107 labeled with electroactive molecule 108 are shown. The linker is a hydrocarbon chain containing 1 to 1000 atoms, which may contain heteroatoms such as O, N, and S. Figure 5 In the example, PEG stands for polyethylene glycol.

[0077] In some embodiments, the systems and methods described herein include nucleotides 107 modified with redox label 108. Free-floating nucleotides added to the sample may include two, three, or four different dNTPs (e.g., dATP, dCTP, dGTP, or dTTP / dUTP). Each type of nucleotide may have a unique redox label. For example, adenine may be modified to include a redox label, and cytosine may be modified to include a different redox label. The sample may include a mixture of labeled and unlabeled nucleotides. For example, dATP, dCTP, and dGTP may each be labeled with a unique redox label, while dTTP / dUTP may be modified with a 3'-ester moiety but without an electroactive label. Alternatively, the apparatus may include more than one well or channel. In one well, the sample may include labeled dTTP and dCTP, wherein the labeling for each nucleotide has a unique electrochemical character. In a second well, the sample may include dATP and dGTP with labels different from each other. Electrochemical measurements from the sample in well 1 can be superimposed with measurements from the sample in well 2 to reconstruct the complete sequence of the polynucleotide chain during post-processing steps. A general example of how DNA chains replicate to incorporate redox-modified nucleotides is provided in U.S. Patent No. 11,131,646, which is incorporated herein by reference in its entirety.

[0078] The systems and methods described herein include enzymes attached to the surface of a dielectric layer. Non-limiting methods for conjugating enzymes to the surface of an electrochemical sensor include: using a bifunctional coupling agent that reacts at one end with a dielectric material such as silica or alumina (e.g., silane chemistry or organophosphoric acid chemistry) and at the other end with a biomolecule (e.g., carboxyl, aldehyde, sulfonic acid, isothiocyanate, NHS ester, epoxide, azalactone, or carbodiimide chemistry); using a double-stranded DNA molecule as a coupling agent between the dielectric layer and the enzyme; physically adsorbing the polymerase onto the dielectric layer rather than covalently attaching it; or using a streptavidin-biotin pair for non-covalent attachment.

[0079] When double-stranded DNA molecules are used as coupling agents between the dielectric layer and the enzyme, oligonucleotides with specific sequences are immobilized onto the dielectric material of the electrochemical sensor. When enzymes modified with oligonucleotides having complementary sequences are introduced, they are immobilized onto the dielectric material via hybridization.

[0080] A key factor in selecting a method for immobilizing proteins onto the surface of a dielectric layer is selecting a method that is more selective for the dielectric material (e.g., alumina) than for the chemistry of the first and second metal electrodes, such that covalent bonding occurs on the dielectric layer between the electrodes rather than on the electrodes themselves.

[0081] During polymerase immobilization, the electric field generated by the first and second electrodes can be used to guide the polymerase to the dielectric layer and induce uniform orientation on the surface. For example, a voltage can be selected to attract the charged polymerase with a symmetrical force, causing the polymerase to bind between the first and second electrodes. When selective adsorption of the polymerase onto the dielectric layer is required, the voltages on the first and second electrodes can be set to generate surface charges that are unfavorable to polymerase attachment to the electrodes (adsorption is reduced when the surface charge matches the isoelectric point of the polymerase). Alternatively, a transverse electric field can be generated to control the orientation of the polymerase molecules, as uniform orientation can lead to improved sensor performance.

[0082] In one embodiment, a protein is attached to the surface of an electrochemical sensor via any of the techniques described above, and then a polynucleotide chain, primers, and redox-modified dNTPs are added to the sensor to form a polynucleotide chain-enzyme-dNTP complex. Sequencing can then be initiated. In an alternative embodiment, the polynucleotide chain, enzyme, and nucleotide reversible terminator (a dNTP modified with a removable blocking group that prevents polynucleotide chain elongation) can be bound together externally to form a polynucleotide chain-enzyme-dNTP complex. The pre-formed complex is then attached to the sensor surface. To initiate sequencing, the blocking group of the nucleotide reversible terminator is released. In one or more embodiments, the blocking group is not an ester group but may be selected from commercially available or novel chemicals. Suitable non-limiting chemicals for the blocking group include 3'-O-allyl, 3'-O-azidomethyl, and 3'-ONH2 groups.

[0083] To accurately determine the sequence of polynucleotide chains using the systems and methods described herein, each electrochemical sensor can only sequence one polynucleotide chain. This can be controlled by providing a low concentration of enzyme during the surface attachment step, such that statistically, each sensor has only 0 or 1 enzyme. Alternatively, the concentration of the target polynucleotide chain can be kept very low compared to the number of available enzymes.

[0084] In other embodiments, a single polynucleotide chain can be preloaded onto a larger entity. For example, a single polynucleotide chain can be preloaded onto a bead of sufficient size to be contained within a well of a sensor containing a plurality of polymerases. Each well can contain only one bead. The bead can be magnetic and therefore can be driven in a magnetic field. Once the bead is in the well, the polynucleotide chain can be released from the bead by an external stimulus such as light, temperature, or pH and attracted directly to the sensor surface via an electric field. Connectors specifically designed to release the polynucleotide chain from the bead upon application of a specific external stimulus can be used to attach the polynucleotide chain to the bead. Unloaded beads can then be released, and the next loaded bead from the preparative library can move into the well.

[0085] In some implementations, when an enzyme-polynucleotide chain-nucleotide complex is formed, the electroactive molecules facilitate electron transfer between the first and second electrodes. Changes in the current signal can be used to identify the identity of the incorporated nucleotide base. To identify each unique incorporated base, each type of nucleotide is modified with a different electroactive label. All four labels can have unique electrochemical properties and can be distinguished from each other based on their current-voltage dependence (IV curves, such as square wave voltammetry or cyclic voltammetry) or based on the amount of current they generate after interacting with the electrodes. Figure 15 Examples of distinguishable electroactive labels and their cyclic voltammetry diagrams are shown. Therefore, it is feasible to modify the four types of nucleotides each with different redox labels. To achieve labels that generate various currents, different numbers of the same electroactive molecules can be attached via linkers with multiple functional groups, i.e., dendritic polymers or polymers.

[0086] In an alternative implementation, three of the four types of nucleotides can be labeled with distinguishable electroactive tags, while the fourth nucleotide is modified with a 3'-ester moiety but lacks an electroactive tag. In this case, the lack of an electrochemical signal can indicate the incorporation of the fourth type of nucleotide. At a minimum, unlabeled nucleotides of two different tagging types can be used for sequence reconstruction.

[0087] In some embodiments, the electrochemical sensor comprises two electrodes separated by a dielectric layer. For each electrode, a voltage V1 is applied to the first electrode, and a voltage V2 is applied to the second electrode. Current then flows from the first and second electrodes respectively to an electronic module designed to sense the current. The current can be positive or negative. When an electroactive molecule enters the sensing region between the first and second electrodes, it can temporarily induce a higher current signal of opposite polarity on each electrode. This is the signal of interest.

[0088] Differential DC current (from the tunneling effect or from current flow via polymerase in the bulk solution) can overload the signal path at the front end. Therefore, the DC gain of the readout electronics can be kept low enough that the output does not rail due to high DC input. Additionally, the AC signal of interest can be amplified above the parasitic DC signal to improve readout.

[0089] Figure 16An embodiment of a readout electronics for an electrode display is shown. This embodiment has three stages. The third stage includes an analog-to-digital (AD) converter that converts an analog voltage signal into a digital readout. The accuracy of the AD converter is set by the signal-to-noise ratio at the input. The function of the first two stages is to separate the DC and AC input signals and amplify the AC signal. The gain of the second stage can be set such that the noise of the AD converter is negligible compared to the amplitude of the AC signal. The first stage sets the voltage of the electrode, which is necessary for oxidation or reduction to occur. The feedback impedance Z1 is selected such that even the largest DC input signal does not overload the output of the first stage. The impedance in the second stage is selected in such a way that only the AC signal is amplified. In an alternative embodiment, Z... 21 It can be purely capacitive. In other implementations, regarding Z... 22 The resistor and capacitor can be parallel. In another implementation, Z 21 The capacitance can be greater than Z. 22 Furthermore, the ratio of the two capacitors can set the gain of the second stage.

[0090] This article describes alternative methods to increase the signal-to-noise ratio to address the problem of high noise levels typically exhibited by electronic devices at lower frequencies. At higher frequencies, the noise floor (i.e., the noise spectral density) is constant. In some implementations, this method includes increasing the signal-to-noise ratio at higher frequencies f... 载波 The operation is performed under conditions where the noise floor is low. For example, the method may include modulation on f 载波 V1 is applied below, and then used around f. 载波 A bandpass filter in a concentrated signal chain. After the bandpass filter, the signal is demodulated back to baseband.

[0091] In various embodiments, the device may include an array of electrically connected sensors.

[0092] In another aspect, a system for nucleic acid sequencing is provided. This system includes at least one device comprising a first electrode, a second electrode, and a dielectric layer disposed between the first and second electrodes. A polymerase is attached to the surface of the dielectric layer. The polymerase incorporates an electroactively labeled dNTP into a polynucleotide chain at the dielectric layer, the electroactive label being covalently bound to the 3'-OH group of the sugar ring of the nucleotide via an ester group, and subsequently cleaves the ester bond linking the electroactive label to the nucleotide to release the free 3'-OH group, allowing for the addition of the next nucleotide. A controller directs the polymerase to be exposed to a sample comprising a polynucleotide chain and an electroactively labeled dNTP, the electroactive label being covalently bound to the 3'-OH group of the sugar ring of the nucleotide via an ester group. Once the polymerase is exposed to the sample comprising the polynucleotide chain and the modified nucleotide, the controller applies a first voltage V1 to the first electrode and a second voltage V2 to the second electrode. The controller then induces the detection of a current flow from each electrode. The detected current can be positive or negative. When an electroactive molecule enters the sensing region between the two electrodes, it temporarily induces a higher current signal of opposite polarity on the two electrodes. The signal from the first electrode can be the inversion of the signal from the second electrode. The controller measures these signals. The controller can calculate the difference between the two current signals. This difference can be used as the final signal for analysis to determine what type of nucleotide is present in the sensing region.

[0093] In principle, conventional targeted or universal library preparation methods can be applied, where the amplicon carries a known flanking sequence representing a universal primer annealing site for a sequencing-by-synthesis reaction. Exemplary methods for preparing template polynucleotide chains include the following three library preparation strategies.

[0094] Standard library preparation: The DNA undergoes blunt-end repair and is tailed with a dA terminator to ligate to a pseudo-double-stranded Y-shaped adaptor with a dT terminator. Amplicon size can range from 50 to 5000 bp. The adaptor can contain a unique molecular identifier to overcome PCR-introduced errors and, where dual redox labeling readout is preferred during sequencing, allows for unique and complete DNA reconstruction. Readout strategies using three or four redox labels are compatible with this library preparation strategy.

[0095] 2D Readout Library Preparation: DNA undergoes blunt-end repair and is tailed with dA to ligate pseudo-double-stranded Y-shaped and hairpin adaptors with dT tails. A positive selection process is then used to obtain the asymmetric ligation products. These products can be used directly for sequencing reactions. Alternatively, amplification can be performed via multiple primer extension or any other isothermal amplification method, including but not limited to loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification, or rITA. Product or amplicon size is determined procedurally by the polymerase used throughout the process. Adaptors may contain unique molecular identifiers to overcome amplification-introduced errors. All readout strategies are compatible with this library preparation strategy. Ligation of sense and antisense strands can increase the accuracy of sequencing readouts.

[0096] Rolling circle library preparation: DNA undergoes blunt end repair and is tailed with a dA terminator to ligate to a hairpin adaptor with a dT terminator. These products can be used directly for sequencing reactions. Alternatively, amplification can be performed via rolling circle amplification (RCA). Product or amplicon size is not limited. The adaptor can contain a unique molecular identifier to overcome errors introduced by amplification. All readout strategies are compatible with this library preparation strategy. The ligation of sense and antisense strands, as well as the multiplicity of such a single locus within a molecule, can increase the accuracy of sequencing readouts.

[0097] Alternatively, a given DNA sample can be sequenced directly using target-specific sequencing primers without prior library preparation. Primer annealing of the prepared DNA can occur on-chip. For example, the prepared DNA can be denatured at 95°C for 1 minute, and then the primers can be allowed to anneal the DNA at 60°C for 1 minute, provided the polymerase can withstand the denaturing conditions. In a preferred embodiment, the primers anneal the prepared DNA strands before loading them onto the chip, and the primer-template DNA conjugate remains stable until it reaches the polymerase.

[0098] Non-restricted polynucleotide chain types that can be sequenced using the systems and methods described herein include DNA and RNA. If RNA is used, it can be upstream processed with reverse transcriptase (RT) to generate cDNA, which can then be read using an immobilized DNA polymerase. Alternatively, the RT enzyme can be immobilized onto the surface of a dielectric layer. When the RNA sequence replicates, the incorporation of redox-modified dNTPs by the RT enzyme can be used to identify the original RNA template. For sequencing random mixtures of RNA molecules, the RNA can be ligated to a universal sequence, which can be used as a docking site for universal primers, at which the sequencing procedure is initiated. Example

[0099] Example 1: Synthesis of a ferrocene-labeled dUTP reversible terminator (A3) with an ester group: Example 1 illustrates a method for synthesizing ferrocene-labeled dUTP reversible terminators with ester groups. The compounds utilized and formed in this method... Figure 6-8 As shown in the image.

[0100] 5'-O-(4,4'-dimethoxytriphenylmethyl)-thymidine-3-O-succinic acid (500 mg, 0.78 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (208 mg, 1.1 mmol), and 4-dimethylaminopyridine (DMAP) (9.5 mg, 0.078 mmol) were dissolved in 5 mL of DCM in a 50 mL round-bottom flask. The mixture was stirred for 10 minutes. Azide-PEG4-amine (244 mg, 1.17 mmol) was dissolved in 1 mL of DCM and added dropwise to the stirred solution under ice-cold conditions. The reaction mixture was allowed to reach room temperature and stirred overnight. As indicated by TLC, the reaction mixture was quenched with 25 mL of 1.0 M HCl once the starting materials were consumed. The entire solution was transferred to a separatory funnel containing 25 mL of DCM, and the organic layer was extracted. The organic layer was washed with another 25 mL of 1.0 M HCl. The organic layer was collected and dried over anhydrous MgSO4. The solution was filtered and the solvent was removed under vacuum. The crude reaction mixture was redissolved in 5 mL of anhydrous DCM. 5% trichloroacetic acid was slowly introduced into the reaction mixture until the solution turned a bright orange color. This is an indication of the deprotection of the dimethoxytriphenylmethyl group, which can be confirmed by TLC. The resulting mixture was evaporated and purified by rapid column chromatography on silica gel (MeOH / CHCl3, 1:10) to obtain compound A1 (190 mg, 45% yield) as a colorless viscous liquid (see example). Figure 6 ).

[0101] At room temperature, a mixture of CuBr (38.7 mg, 0.270 mmol) and trihydroxypropyltriazolylmethylamine (THPTA) (118 mg, 0.272 mmol) in 2 mL of acetonitrile (ACN) was slowly added to a solution of compound 2 (50 mg, 0.092 mmol) and acetylenylferrocene (23 mg, 0.109 mmol) in 1 mL of acetonitrile (ACN). The reaction mixture was stirred overnight until all of compound 2 was consumed as indicated by TLC. The organic solvent was evaporated, and the crude mixture was washed with 25 mL of aqueous solution. Extraction was performed using 2 x 25 mL of ethyl acetate. The organic layers were combined and dried over anhydrous MgSO4. The solvent was removed under vacuum, and the crude mixture was purified by rapid column chromatography on silica gel (methanol-chloroform, 0.5:10) to obtain compound A2 (27 mg, 46% yield) as a yellow viscous liquid. See example Figure 7 ).

[0102] Compound 3 (25 mg, 0.033 mmol) and 1,8-bis(dimethylamino)naphthalene (DMAN) (7.2 mg, 0.034 mmol) were measured in a 50 mL round-bottom flask, and tributylammonium pyrophosphate (TBAP) (17 mg, 0.031 mmol) was weighed in a separate vial. Both were dried under high vacuum on P2O5 (approximately 500 mg) for 1 hour. Tributylamine (NBu3) (0.05 mL, 0.210 mmol) was cooled to -20 °C. After the drying process, three vacuum-nitrogen cycles were performed on both the flask and the vial. 0.5 mL of acetonitrile was added to the vial containing TBAP and cooled to -20 °C. Then, 0.5 mL of trimethyl phosphate was slowly added to the flask containing compound 3 and DMAN, and the flask was continuously stirred for 10 minutes under a mixture of ice and dry ice. Phosphorus oxychloride was then added dropwise to the flask over a 5-minute period, and stirred for another 30 minutes in an ice / dry ice bath. Cooled NBu3 and TBAP were then added to the reaction mixture, and the mixture was stirred for another hour under ice-cold conditions. 1.0 M cooled TEAB buffer was introduced into the reaction mixture, and the mixture was stirred for another hour. The solution was concentrated under vacuum to remove any residual organic solvent. The resulting aqueous solution of the reaction mixture was frozen at -80°C for lyophilization. The frozen mixture was lyophilized and purified using reversed-phase HPLC (C-18 column, TEAB / acetonitrile linear gradient) to give compound A3 (see example, Figure 8 ).

[0103] Example 2: Synthesis of a ferrocene-labeled dUTP reversible terminator (B5) with an ester group. The compound utilized and formed in this method... Figure 9-13 As shown in the image.

[0104] Sodium azide (1.3 g, 20 mmol) was placed in a 100 mL double-necked round-bottom flask, and oxygen was removed from the reaction environment by three cycles of vacuum / argon purging. 20 mL of acetonitrile was added to the flask, and the mixture was cooled in an ice bath for 10 min. Then, thioyl chloride (1.6 mL, 20 mmol) was injected into the flask over a 2-minute period. The solution was allowed to return to room temperature. After 12 hours, the reaction flask was cooled in an ice bath for 10 min. Then, imidazole (2.6 g, 40 mmol) was added aliquots to the reaction mixture. The flask was warmed to room temperature, and stirring was continued for another 6 hours. 50 mL of ethyl acetate was added to the reaction mixture, and the entire solution was transferred to a separatory funnel. The solution was washed with 50 mL of water, followed by 50 mL of saturated NaHCO3 solution, and finally with 50 mL of brine solution. The organic layer was collected and cooled in an ice bath. An ethanolic solution of HCl was prepared by dissolving 2.1 mL of CH3COCl in 7.5 mL of ice-cold anhydrous ethanol. The HCl solution was cooled in an ice bath for another 10 minutes and then added dropwise to the organic layer with stirring. The product, imidazole sulfonyl azide, appeared as a white precipitate, and the suspension was cooled again and filtered under vacuum using a Hirsch funnel to obtain compound B1 (2 g, 58% yield) (see example). Figure 9 ).

[0105] Imidazole sulfonyl azide (38 mg, 0.22 mmol) and amine-(PEG)4-acid (50 mg, 0.19 mmol) were dissolved in 1 mL of methanol in a 25 mL round-bottom flask. K₂CO₃ was then added to the reaction mixture, followed by the addition of 0.2 mL of a CuSO₄·5H₂O solution in methanol. The solution initially turned blue and gradually turned yellow over a period of 1 hour. The solution was then stirred overnight at room temperature. 5 mL of methanol was added to the reaction mixture, and the solution was filtered using gravity filtration. The solution was then evaporated to remove the solvent. The resulting crude mixture was used directly for the next step without further purification.

[0106] At room temperature, a mixture of CuBr (33 mg, 0.23 mmol) and trihydroxypropyltriazolylmethylamine (THPTA) (100 mg, 0.23 mmol) in 2 mL of acetonitrile (ACN) was slowly added to a solution of the crude mixture from the previous step (approximately 55 mg, 0.19 mmol) and acetylenylferrocene (50 mg, 0.24 mmol) in 1 mL of acetonitrile (ACN). The reaction mixture was then stirred overnight until all of compound 2 was consumed as indicated by TLC. The organic solvent was then evaporated, and the crude mixture was washed with 25 mL of aqueous solution and extracted with 2 x 25 mL of ethyl acetate. The organic layers were combined and dried over anhydrous MgSO4. The solvent was then removed under vacuum, and the crude mixture was purified by rapid column chromatography (water / acetonitrile, 1:5) on a reversed-phase C-18 column to obtain compound B2 (23 mg, 24% yield) as a yellow viscous liquid (see example). Figure 10 ).

[0107] 100 mg of thymidine (0.41 mmol) and 60 mg of imidazole (0.88 mmol) were charged into a 50 mL double-necked round-bottom flask and kept under vacuum for 10 min. 1 mL of anhydrous DMF was then added to the flask under argon conditions, and stirring was maintained for 10 min. 65 mg of tert-butyldimethylchlorosilane (TBDMSCl) (0.43 mmol) was added aliquoted through one neck of the flask while the other neck was kept under a continuous argon flow. The reaction was stirred overnight at room temperature. Once the thymidine, as indicated by TLC, was completely consumed, the reaction was quenched by adding 25 mL of DI water. A white precipitate of the product was observed upon quenching. The solution was then washed with 2 x 25 mL of ethyl acetate. The resulting ethyl acetate solutions were combined and dried over anhydrous MgSO4. The solution was filtered, and the ethyl acetate was removed under vacuum. The resulting crude mixture was purified by rapid column chromatography (methanol / chloroform 1:10) on a basic alumina gel to obtain compound B3 as a white powder. (112 mg, yield 72%) (see example) Figure 11 ).

[0108] 5'-O-tert-butyldimethylsilylthymidine (20 mg, 0.054 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (14 mg, 0.073 mmol), and 4-dimethylaminopyridine (DMAP) (1 mg, 0.008 mmol) were dissolved in 5 mL of DCM in a 50 mL round-bottom flask. The solution was stirred for 10 minutes. Compound 3 (23 mg, 0.046 mmol) was dissolved in 1 mL of DCM and added dropwise to the stirred solution under ice-cold conditions. The reaction mixture was allowed to reach room temperature and stirred overnight. As indicated by TLC, the reaction mixture was quenched with 25 mL of 1.0 M HCl once the starting material was consumed. The entire solution was transferred to a separatory funnel containing 25 mL of DCM, and the organic layer was extracted. The organic layer was washed with another 25 mL of 1.0 M HCl. The organic layer was collected and dried using anhydrous MgSO4. The solution was filtered, and the solvent was removed under vacuum. The resulting crude mixture was purified by rapid column chromatography on silica gel (MeOH / CHCl3, 1:10) to obtain the intermediate compound (5'-O-tert-butyldimethylsilylthymidine-PEG4-ferrocene). The obtained product was redissolved in 1 mL of anhydrous THF. A solution of 1.0 M tetrabutylammonium fluoride (TBAF) (0.2 mL) in THF was added dropwise to the above solution, and stirring was maintained for 30 min. After the reaction was complete as indicated by TLC, the solvent was evaporated, followed by the addition of 20 mL of saturated NaHCO3 solution. The solution was extracted with 2 x 20 mL of ethyl acetate. The organic layers were combined and dried using anhydrous MgSO4. The solution was filtered, and the solvent was removed to obtain compound B4 (7 mg, 21% yield) as a yellow viscous liquid (see example). Figure 12 ).

[0109] Compound B5 was obtained via triphosphorylation of B4 by following the same experimental procedure as compound A3 (see example). Figure 13 ).

[0110] Example 3: Synthesis of a ferrocene-labeled reversible terminator (C3) with an ester group and no linker.

[0111] like Figure 14 Example 3, shown in the figure, illustrates a method for synthesizing a ferrocene-labeled reversible terminator (C3) having an ester group and no linker.

[0112] Compound C3 was synthesized using a procedure similar to that described in Examples 1 and 2. Compound B3 (0.31 g, 0.87 mmol), ferrocene carboxylic acid (0.1 g, 0.435 mmol), EDC (0.092 g, 0.048 mmol), and DMAP (0.0026 g, 0.021 mmol) were dissolved in 12 mL of anhydrous DCM under an anaerobic atmosphere and stirred at room temperature. The conversion of compound B3 was monitored by TLC using hexane / ethyl acetate as the eluent. After the reaction was complete, the solvent was evaporated, and the product was purified by rapid silica gel column chromatography with a linear gradient of hexane / ethyl acetate. Yield of the isolated product: 0.06 g. Compound C1 was deprotected to obtain compound C2 by following the same procedure as for compound B4. The final compound C3 was obtained after a triphosphorylation procedure similar to that used for the synthesis of compounds A3 and B5.

[0113] While exemplary embodiments have been described above, it is not intended that these embodiments describe all possible forms covered by the claims. The language used in this specification is descriptive rather than restrictive, and it should be understood that various changes can be made without departing from the spirit and scope of this disclosure. As previously stated, features of the various embodiments can be combined to form further embodiments of this disclosure, which may not be explicitly described or shown. Although various embodiments may be described as providing an advantage or preference with respect to one or more desired characteristics over other embodiments or prior art implementations, those skilled in the art will recognize that one or more features or characteristics can be compromised to achieve desired overall system properties, depending on the specific application and implementation. These properties may include, but are not limited to, cost, strength, durability, lifecycle cost, marketability, appearance, packaging, size, maintainability, weight, manufacturability, ease of assembly, etc. Therefore, any embodiment described as being less desirable than other embodiments or prior art implementations in terms of one or more characteristics is not departing from the scope of this disclosure and may be desirable for a particular application.

Claims

1. A method for nucleic acid sequencing, comprising: Provide at least one device comprising: A first electrode, a second electrode, and a dielectric layer defining a sensing region between the first electrode and the second electrode; and A polymerase attached to the surface of the dielectric layer, wherein the polymerase targets polynucleotide chains to the dielectric layer; The at least one device is provided with a sample comprising a polynucleotide chain and at least one modified nucleotide having an electroactive label covalently bound to the 3'-OH of the sugar ring of the nucleotide via an ester group; A first potential is applied to a first electrode and a second potential is applied to a second electrode to induce an electron flow between the first and second electrodes, thereby generating a measurable electrical signal when an electroactive label is present in the sensing region; and A first signal from the first electrode and a second signal from the second electrode are detected to determine when the modified nucleotide is present in the sensing region.

2. The method of claim 1, wherein the polymerase incorporates the modified nucleotide into the polynucleotide chain, the modified nucleotide having an electroactive label covalently bound to the 3'-OH of the sugar ring of the nucleotide via an ester group.

3. The method of claim 2, wherein the polymerase cleaves the ester group of the modified nucleotide after nucleotide incorporation to remove the electroactive label from the sensing region.

4. The method according to claim 3, wherein the polymerase is one of a group A polymerase or a group B polymerase.

5. The method of claim 1, wherein two or more of dATP, dCTP, dGTP, or dTTP / dUTP are modified with measurably different electroactive labels.

6. The method of claim 1, wherein the modified nucleotide having an electroactive label covalently bound to the 3'-OH of the sugar ring of the nucleotide via an ester group has the following formula: in: X is It's a single button, a double button, and a triple button. The bases are adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). L (connector) is either absent or contains a hydrocarbon chain of 1 to 1000 atoms, and may contain heteroatoms such as O, N, and S. n = 1-1000， R1 is H or OH, and R2 is a redox marker.

7. The method of claim 1, further comprising calculating the difference between the first signal and the second signal to determine when the modified nucleotide is present in the sensing region.

8. A system for nucleic acid sequencing, comprising: At least one device comprising: At least one electrochemical sensor includes a first electrode, a second electrode, and a dielectric layer defining a sensing region between the first and second electrodes; and a polymerase attached to the surface of the dielectric layer, wherein the polymerase targets polynucleotide chains to the sensing region; and The controller is configured as follows: A first current is directed through a first electrode and a second current is directed through a second electrode to induce an electron flow between the first and second electrodes, thereby generating a measurable electrical signal when an electroactive label is present in the sensing area. The at least one device is provided with a sample comprising a polynucleotide chain and at least one modified nucleotide, the modified nucleotide having an electroactive label covalently bound to the 3'-OH group of the nucleotide's sugar ring via an ester group; and The first current flowing out of the first electrode and the second current flowing out of the second electrode are detected.

9. The system of claim 8, wherein the polymerase incorporates a modified nucleotide into a polynucleotide chain, the modified nucleotide having an electroactive label covalently bound to the 3'-OH of the sugar ring of the nucleotide via an ester group, and wherein the polymerase cleaves the ester group of the modified nucleotide after incorporation to remove the electroactive label from the sensing region.

10. The system of claim 9, wherein the polymerase is one of a group A polymerase or a group B polymerase.

11. The system of claim 8, wherein a measurable electrical signal is generated while the electroactive label is still incorporated into the enzyme-template-dNTP complex and while the electroactive label is located within the sensing region.

12. The system of claim 8, wherein the controller is further configured to calculate the difference between the first current and the second current to determine when the modified nucleotide is present in the sensing region.

13. The system of claim 8, wherein the modified nucleotide having an electroactive label covalently bound to the 3'-OH of the sugar ring of the nucleotide via an ester group has the following formula: in: X is It's a single button, a double button, and a triple button. The bases are adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). L (connector) is either absent or contains a hydrocarbon chain of 1 to 1000 atoms, and may contain heteroatoms such as O, N, and S. n = 1-1000， R1 is H or OH, and R2 is a redox marker.

14. The system according to claim 8, wherein the at least one device is a plurality of devices.

15. A method for forming a system for nucleic acid sequencing, the method comprising: Provide a first electrode and a second electrode; A dielectric layer is placed between the first electrode and the second electrode to define the sensing area; A polymerase is attached to the surface of the dielectric layer, wherein the polymerase incorporates modified nucleotides into the polynucleotide chain, the modified nucleotides having an electroactive label covalently bound to the 3'-OH group of the nucleotide's sugar ring via an ester group; and Electrodes are configured to detect changes in current when an electroactive label is present in the sensing area.

16. The method of claim 15, further comprising generating two of dATP, dCTP, dGTP, and dTTP / dUTP, each of which is modified with a measurably different electroactive label, the electroactive label being covalently bound via an ester group to the 3'-OH of the sugar ring of dATP, dCTP, dGTP, and dTTP / dUTP.

17. The method of claim 15, further comprising generating dATP, dCTP, dGTP, and dTTP / dUTP, each modified with a measurably different electroactive label, the electroactive label being covalently bound via an ester group to the 3'-OH of the sugar ring of dATP, dCTP, dGTP, and dTTP / dUTP.

18. The method of claim 15, wherein the polymerase cleaves the ester group of the modified nucleotide after nucleotide incorporation to remove the electroactive label from the sensing region.

19. The method of claim 15, further comprising configuring the controller to: A first current is guided through the first electrode and a second current is guided through the second electrode to induce an electron flow between the first and second electrodes; The polymerase is directed to be exposed to a sample comprising a polynucleotide chain and dNTPs modified with an electroactive label, the electroactive label being covalently bound via an ester group to the 3'-OH group of the sugar ring of the nucleotide; and The first current flowing out of the first electrode and the second current flowing out of the second electrode are detected.

20. The method of claim 19, wherein the controller is further configured to calculate the difference between the first current and the second current to determine when the modified nucleotide is present in the sensing region.

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