Nucleic acid detection using nanogap electrical sensors

Abstract

Systems and methods for detecting polynucleotide strands in a sample during an early stage of an amplification cycle. A metal-insulator-metal (MIM) electrode unit having two metal electrodes separated by a dielectric layer may be used as a nanogap sensor. Primers for amplifying a target polynucleotide strand may be immobilized on the surface of the dielectric layer. Polymerase enzymes, nucleotides, templates, and primers may be added to a sample. At least one type of nucleotide may be labeled with a redox label that may enhance the transport of electrons through the nanogap via a tunnel and / or diffusion-based redox cycle. A signal (e.g., a voltage pulse) may be directed through each electrode. A current value from each electrode may be measured to establish a baseline signal. A change in the baseline signal may indicate the presence of a target polynucleotide strand. The time to detect the change in the baseline signal may facilitate the quantification of the target polynucleotide strand.

Description

Technical Field

[0001] The present disclosure relates to systems, devices, and methods for detecting polynucleotide chains in a sample. Background Art

[0002] Molecular diagnostic tools that rely on nucleic acid amplification technology primarily use detection systems that rely primarily on cumbersome and expensive optical measurements. Nanogap-based electrical measurement of current via tunneling and electrochemical redox methods may provide a faster and more cost-effective detection mechanism. When redox tag molecules reach the active sensing zone near the two electrodes, the nanogap electrodes can measure the increased tunneling current. In addition, the redox tag, which diffuses back and forth multiple times between the two electrodes, also induces a redox current. Summary of the Invention

[0003] In at least one aspect, a method for detecting a polynucleotide chain in a sample is provided. The method may include providing a device comprising a nanogap sensor, the nanogap sensor comprising a first electrode and a second electrode separated by a dielectric layer, wherein a primer is attached to the surface of the dielectric layer. The method may additionally include providing a sample to the device, wherein the sample is a solution comprising a primer, a polymerase, a target polynucleotide chain, and at least one nucleotide modified with a redox tag. The method may further include transmitting a first signal to the first electrode to generate a first current value; transmitting a second signal to the second electrode to generate a second current value; measuring the first current value and the second current value to determine a baseline signal; and measuring a change in the baseline signal to detect the presence of a target polynucleotide chain.

[0004] In another aspect, a method for detecting a polynucleotide chain in a sample is provided. The method may include providing a device including a nanogap sensor, the nanogap sensor including a first electrode, a dielectric layer arranged on the first electrode, and a second electrode arranged on the dielectric layer to form an electrode stack (electrode stack), wherein the electrode stack includes a well compartment (well compartment) of a size suitable for receiving a sample solution, and wherein the dielectric layer includes a nanocavity to which a primer is attached. The method may additionally include providing a sample solution to the device, wherein the sample solution includes a primer, a polymerase, a target polynucleotide chain, and at least one nucleotide modified with a redox tag. The method may further include delivering a first signal to the first electrode to generate a first current value, and delivering a second signal to the second electrode to generate a second current value; measuring the first current value and the second current value to determine a baseline signal; and measuring a change in the baseline signal to detect the presence of a target polynucleotide chain.

[0005] In yet another aspect, a system for detecting a polynucleotide chain in a sample is provided. The system may include a device comprising a nanogap sensor, the nanogap sensor comprising a first electrode, a dielectric layer disposed on the first electrode, and a second electrode disposed on the dielectric layer to form an electrode stack, wherein the electrode stack comprises a pore compartment sized to receive a sample solution, wherein the sample solution comprises a primer, a polymerase, a target polynucleotide chain, and at least one nucleotide modified with a redox tag, and wherein the primer is attached to the dielectric layer in fluid communication with the sample solution. The system may further include a controller configured to, when the sample solution is in the device, transmit a first signal to the first electrode to generate a first current value, and transmit a second signal to the second electrode to generate a second current value; measure the first current value and the second current value to determine a baseline signal; and measure a change in the baseline signal to detect the presence of a target polynucleotide chain. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1A and 1B Schematic side views and schematic top views are shown, respectively, of a metal-insulator-metal electrode device with a nanogap according to an embodiment.

[0007] Figure 2 A schematic diagram showing the composition of a sample solution and the structure of a metal-insulator-metal electrode according to an embodiment is shown.

[0008] Figure 3 Shown is a schematic diagram of a signaling mechanism for detecting a target polynucleotide strand according to at least some embodiments.

[0009] Figures 4A-4F A schematic diagram showing a signal detection scenario of a sample solution having a low concentration of target polynucleotide chains according to an embodiment is shown.

[0010] Figures 5A-5D A schematic diagram showing a signal detection situation of a sample solution having a high concentration of target polynucleotide chains according to the embodiment is shown.

[0011] Figure 6 A schematic diagram of an electrical measurement configuration according to an embodiment is shown.

[0012] Figure 7A and 7B An example of a voltage distribution applied to both the first electrode and the second electrode according to at least one embodiment is shown.

[0013] Figure 8 A schematic diagram of a nanogap undercut between first and second electrodes is shown.

[0014] Figure 9A and 9B Schematic side and top views, respectively, are shown of examples of potential nanogap sensing device designs according to embodiments.

[0015] Figures 10A-10B A side view and a top view of a nanogap sensing device with an oval-shaped window opening are shown, respectively. Figures 10C-10D respectively show polygonal shapes ( Figure 10B ) Side and top views of the nanogap sensing device with a window opening.

[0016] Figures 11A-11B The side view and top view of a nanogap sensing device with an oval-shaped window opening are shown, respectively, where the first electrode (metal electrode 1) is not completely etched away. Figures 11C-11D The side view and top view of a nanogap sensing device with a polygonal-shaped window opening are shown, respectively, where the first electrode (metal electrode 1) is not completely etched away.

[0017] Figure 12A and 12B Schematic side view and schematic top view of an example of a nanogap sensing device are shown, respectively, where the shape of the sensing region is in the form of a single line.

[0018] Figure 13A and 13B Schematic side and top views, respectively, of an example nanogap sensing device are shown, where the electrodes are flattened and the active sensing components face upward.

[0019] Figure 14 A schematic example of an array format of nanogap sensors for multiplexed testing is shown.

[0020] Figures 15A-15J Shown are examples of suitable modified dNTPs (labeled with a ferrocene redox label) that can be enzymatically incorporated into a polynucleotide chain.

[0021] Figures 16A-16C Examples of suitable modified dNTPs (labeled with an anthraquinone redox label) that can be enzymatically incorporated into a polynucleotide chain are shown.

[0022] Figures 17A-17B Shown are examples of suitable modified dNTPs (labeled with a methylene blue redox label) that can be enzymatically incorporated into a polynucleotide chain.

[0023] Figures 18A-18CExamples of suitable modified dNTPs (labeled with a phenothiazine redox label) that can be enzymatically incorporated into a polynucleotide chain are shown. Detailed description

[0024] As required, detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be implemented in various and alternative forms. The drawings are not necessarily to scale; some features may be exaggerated or minimized to show details of particular components. Therefore, the specific structural and functional details disclosed herein should not be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to employ the present invention in various ways.

[0025] Except in the examples, or where otherwise expressly indicated, all numerical quantities in this specification indicating amounts of materials or conditions of reaction and / or use are to be understood as modified by the word "about". The first definition of an acronym or other abbreviation applies to all subsequent uses of the same abbreviation herein and, mutatis mutandis, to conventional grammatical variations of the initially defined abbreviation; and, unless expressly indicated otherwise, measurements of properties are determined by the same techniques as previously or subsequently cited for the same property.

[0026] Unless defined otherwise, 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.

[0027] It is also to 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, vary. Furthermore, the terminology used herein is for describing specific embodiments only and is not intended to be limiting in any way.

[0028] It must also be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a component mentioned in the singular is intended to include a plurality of components.

[0029] The terms "or" and "and" are used interchangeably and may be understood to mean "and / or".

[0030] The term "comprising" is synonymous with "including," "having," "containing," or "characterized by." These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.

[0031] The phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When the phrase appears in a clause of the claim body, rather than immediately after the preamble, it limits only the elements specified in that clause; the claim as a whole does not exclude other elements.

[0032] The phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps, and to those materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0033] The terms "comprising," "consisting of," and "consisting essentially of are used interchangeably. When one of these three terms is used, the presently disclosed and claimed subject matter may include the use of either of the other two terms.

[0034] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably in this disclosure. They may refer to nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or a polymeric form of their analogs. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. 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 comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides suitable for use in the described embodiments. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. 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.

[0035] The term "complementarity" or "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence by traditional Watson-Crick or other non-traditional types. The complementarity percentage represents 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, and 6 out of 6 are 66.67%, 83.33%, and 100% complementary, respectively). "Fully complementary" can mean that all consecutive residues of a nucleic acid sequence can form hydrogen bonds with the same number of consecutive residues in a 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% over a region of more than 4, 5, 6, 7, and 8 nucleotides, or a percentage therebetween, or refers to two nucleic acids that hybridize under stringent conditions.

[0036] As used herein, the term "enzyme" or "polymerase" may refer to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein capable of transferring a polynucleotide chain. Non-limiting examples of proteins capable of transferring a polynucleotide chain include DNA polymerases, RNA polymerases, ribosomes, single-stranded 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 expressly stated, all R groups (e.g., R i , wherein i is an integer) includes 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 6-18Aryl; a 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", wherein R', R" and R"' are C 1-10 Alkyl or C 6-18 aromatic groups; indication of a moiety or structure with a positive charge implies the presence of one or more negative counterions to balance the charge, and similarly, indication of a moiety or structure with a negative charge implies the presence of one or more positive counterions to balance the charge; percentages, "parts of" and ratio values ​​are all by weight; the term "polymer" includes "oligomers," "copolymers," "terpolymers," and the like; molecular weights provided for any polymer may refer to weight average molecular weight unless otherwise indicated; a description of a group or class of materials as suitable for a given purpose in connection with one or more embodiments may mean that mixtures of any two or more members of the group or class are also suitable; descriptions of ingredients in chemical terms refer to the ingredients when added to any combination specified in the specification and do not necessarily preclude chemical interactions between the ingredients of the mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses of the same abbreviation in this document and, mutatis mutandis, to conventional grammatical variations of the initially defined abbreviation; and, unless expressly stated otherwise, measurements of properties are determined by the same techniques as previously or subsequently cited for the same property.

[0038] As used herein, the term "alkyl" may refer to a C 1-20The term "lower alkyl" refers to an alkyl radical having 1 to about 8 carbon atoms (i.e., C1-C2). 1-8 "alkyl" refers to an alkyl group having from about 10 to about 20 carbon atoms, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. "Higher alkyl" refers to an alkyl group having from about 10 to about 20 carbon atoms, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. "Higher alkyl" refers to an alkyl group having from about 10 to about 20 carbon atoms, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.

[0039] As used herein, the term "aryl" may refer to an aromatic substituent, which may be a single aromatic ring, or multiple aromatic rings fused together, covalently linked or connected to a common group (such as, but not limited to, a methylene or ethylene moiety). Common linking groups may also be carbonyl (such as in benzophenone) or oxygen (such as in diphenyl ether). Examples of aryl include, but are not limited to, phenyl, naphthyl, biphenyl and diphenyl ether. Aryl includes heteroaryl, wherein one or more aromatic rings contain heteroatoms (such as N, O, S or Se). Exemplary heteroaryl groups include, but are not limited to, furyl, pyridyl, pyrimidyl, imidazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, thienyl, etc. An aryl group can be optionally substituted with one or more aryl substituents ("substituted aryl") which can be the same or different, wherein "aryl substituents" include 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, hydroxy, acyl, carboxyl, alkoxy (e.g., methoxy), aryloxy, aralkyloxy, thioalkyl, thioaryl, thioaralkyl, amino (e.g., aminoalkyl, aminodialkyl, aminoaryl, etc.), sulfonyl, and sulfinyl.

[0040] It should also be understood that the integer ranges clearly include all integers therebetween. For example, the integer range 1-10 clearly 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 needed, the digits therebetween can be used as alternative upper or lower limits, the digits therebetween being the difference between the upper and lower limits divided by an increment of 10. For example, if the range is 1.1 to 2.1, the following digits 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0 can be selected as lower or upper limits. In the specific embodiments proposed herein, concentration, temperature and reaction conditions (such as pressure, pH, etc.) can be implemented with plus or minus 50% of the specified value rounded to three significant figures. In a refinement, the concentrations, temperatures, and reaction conditions (e.g., pressure, pH, etc.) may be implemented with plus or minus 30% of the specified value rounded to three significant figures of the values ​​provided in the examples. In another refinement, the concentrations, temperatures, and reaction conditions (e.g., pressure, pH, etc.) may be implemented with plus or minus 10% of the specified value rounded to three significant figures of the values ​​provided in the examples.

[0041] In the examples described herein, the concentrations, temperatures, and reaction conditions (e.g., pressure, pH, flow rate, etc.) may be implemented with plus or minus 50% of the specified value rounded or truncated to two significant figures of the values ​​provided in the examples. In a refinement, the concentrations, temperatures, and reaction conditions (e.g., pressure, pH, flow rate, etc.) may be implemented with plus or minus 30% of the specified value rounded or truncated to two significant figures of the values ​​provided in the examples. In another refinement, the concentrations, temperatures, and reaction conditions (e.g., pressure, pH, flow rate, etc.) may be implemented with plus or minus 10% of the specified value rounded or truncated to two significant figures of the values ​​provided in the examples.

[0042] In the present application, electroactive molecules include redox molecules, and redox signals include electrical signals such as baseline current changes, which can be visualized as spikes or fluctuations. Polynucleic acids (NA) or polynucleotide chains include DNA, and nucleotides include dNTPs.

[0043] The processes, methods, or algorithms disclosed herein may be delivered to or implemented by a processing device, a controller, or a computer, which may include any existing programmable electronic control unit or a dedicated electronic control unit. Similarly, the processes, methods, or algorithms may be stored as various forms of data and instructions that can be executed by a controller or a computer. The processes, methods, or algorithms may also be implemented in executable software objects. Alternatively, 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, may be used to implement the processes, methods, or algorithms in whole or in part.

[0044] Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

[0045] The term molecular diagnostics encompasses a class of tests that identify biomarkers. For example, molecular diagnostic tests can identify genetic DNA mutations that cause disease. Molecular diagnostics can also identify the presence of DNA, RNA, or protein in a sample (such as a human blood sample) for the diagnosis of conditions including, but not limited to, diseases, allergies, or autoimmune conditions.

[0046] Given the usefulness of molecular diagnostics in helping to combat highly contagious diseases such as SARS-CoV-2, they are receiving increasing mainstream attention. In addition to identifying viral RNA in human samples to diagnose and map infections, molecular diagnostic tests have also been influential in tracking the lineage of diseases such as SARS-CoV-2 by identifying changes in viral RNA sequences. As a result, such diseases have greatly increased the demand for rapid tests that can be performed on-site and that can process a large number of test samples with high sensitivity and selectivity at a reasonable cost. Therefore, there is a need for technological advancements over currently available solutions.

[0047] Various molecular diagnostic applications have utilized electrical detection using nanogap-based structures. This type of system may be very sensitive to changes in or near the nanogap, making it highly useful in real-world detection situations. The high sensitivity conferred by this system can allow detection of biomolecules present in lower concentrations in a sample. In this way, the diagnosis of a disease or condition can be achieved earlier after infection or onset. In addition, the electrical detection mechanism does not require a bulky system, such as the system typically used for optical-based detection. The high sensitivity that can be achieved with nanogap-based sensors also greatly reduces the time required to complete the test. Therefore, faster detection can be achieved with simpler hardware, thereby generating the potential for portable, high-throughput measurement systems. These benefits collectively provide benefits for point-of-care applications.

[0048] Many molecular diagnostic tools (e.g., those designed to identify patients infected with diseases such as SARS-CoV-2) rely on nucleic acid amplification technology. Although nucleic acid amplification technologies such as transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HAD), and recombinase polymerase amplification (RPA) have improved over previous technologies, their detection still relies primarily on cumbersome and expensive optical measurements, given the reduced amplification times they offer.

[0049] Nanogap-based electrical measurement of current via tunneling and electrochemical redox methods could offer a faster and more cost-effective detection mechanism. First, the nanogap electrodes measure the increased tunneling current when redox tag molecules reach the active sensing region near two electrodes. Second, the redox tag, which diffuses back and forth multiple times between the two electrodes, also induces a redox current. This requires a system that can be effectively used at the point of care to quickly and accurately identify nucleic acids of interest in patient samples in a cost-effective manner.

[0050] In one or more embodiments, the present invention provides a system and method for detecting nucleic acid during the early stage of the amplification cycle. The system and method described herein can sense the nucleic acid target molecule in the solution phase by measuring the current change of the electrode pair and the nanogap dielectric material therebetween during the early stage of amplification. According to various embodiments, a metal-insulator-metal (MIM) electrode unit can be used as a sensor, wherein the first metal electrode is separated from the second metal electrode by a nanogap dielectric. Primers for nucleic acid amplification can be fixed on the surface of a thin dielectric nanogap material. In order to amplify, polymerase enzymes, dNTPs, templates and primers can be added to the sample solution, and at least one type of nucleotide can be labeled with a redox tag, which can enhance the transmission of electrons through the nanogap via a tunnel or a diffusion-based redox cycle. Amplified nucleic acids containing nucleotides modified with redox tags can be combined with fixed primers on the nanogap. The presence of the redox tag can induce changes in the electron transfer between the first and second electrodes, which can be detected in the form of spikes, baseline current changes or fluctuations. When the template binds to the immobilized primer and the nucleotide modified with the redox tag is enzymatically incorporated into the nucleic acid copy by the polymerase, changes in electron transfer can be similarly detected. Because the presence of even a single molecule can induce a change in the current of the nanoconfined structure, detection can occur during the early stages of amplification, which can reduce detection time in biosensing applications.

[0051] Figure 1AA schematic side view of a nanogap electrode sensor device "device" 100 according to an embodiment is shown. Device 100 may include a substrate 102 and a dielectric bottom 104 at least partially disposed on substrate 102. Device 100 may additionally include a first electrode 106 at least partially disposed on dielectric bottom 104. The device may additionally include a dielectric layer 108 at least partially disposed on first electrode 106. The dielectric layer may also be referred to as a nanogap or gap dielectric. A second electrode 110 may be at least partially disposed on dielectric layer 108. A dielectric cap 112 may be at least partially disposed on the second electrode. An active sensing zone 111 may be formed by etching an electrode stack in the form of a window. The sensing zone may be disposed along the perimeter of the window. As shown in FIG. 1 , a rectangular window shape is shown. It should be appreciated that the shape of the window etched into the electrode stack may be any shape, and the shape may be selected based on its specific effect on the steps of the method as described herein. Since the window opening can be used as a well compartment 113 for reagent reactions, the shape of the window may affect parameters including but not limited to filling, washing, and flowing solutions. Depending on the properties of the surface and the composition of the sample solution, the design can be optimized to avoid problems such as bubble capture or crosstalk with adjacent wells (e.g., in the case of multiplexing).

[0052] The format of the electrode can be flexible, provided that the distance between the electrodes is accurately set. The electrode and the nanogap can be formed by a material that is stable in the test solution under multiple rounds of electrochemical measurements, and is compatible with the surface modification for fixing the primer. The electrode material can include but is not limited to platinum, gold, titanium nitride and palladium or conductive graphene. The dielectric material can include but is not limited to ceramics (including 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 or dielectric graphene. The thickness of the nanogap dielectric can be less than 20nm.

[0053] Figure 2Shown is a device 100 exposed to a sample solution 114 according to an embodiment. The sample solution 114 can include nucleotides (also referred to as dNTPs 116), primers 118, enzymes 120, and target polynucleotide chains 122. The sample solution 114 can also include a buffer and salt. The sample solution can be referred to interchangeably as a "sample solution" or a "bulk solution". The enzyme 120 can be a polymerase that can incorporate dNTPs into a polynucleotide chain. In some embodiments, primers 118 can float freely in the sample solution 114. Primers 118b can also be fixed to the surface of the gap dielectric 108. In at least some embodiments, some primers 118 can float freely in the sample solution, while other primers 118b can be fixed to the surface of the gap dielectric 108. In this way, the amplification of the target polynucleotide chain 122 can occur simultaneously in the bulk solution 114 and on the surface of the gap dielectric 108. For example, a target polynucleotide chain 122 floating in the bulk solution 114 can bind to a primer 118 floating in the bulk liquid 114, and amplification of the bound target polynucleotide chain 122 can occur. Simultaneously, other target polynucleotide chains 122 floating in the bulk solution can bind to a primer 118b attached to the dielectric layer 108, and the bound target polynucleotide chain 112 can be amplified on the surface of the dielectric layer 108. The target polynucleotide chain 122 bound to the primer 118b attached to the dielectric layer 108 may or may not contain a dNTP modified with a redox tag. During amplification and detection of the target polynucleotide chain, the same sample solution 114 having the primer 118, the polymerase 120, the target polynucleotide chain 122, and the dNTP 116 (at least one type of dNTP modified with a redox tag) can remain in the well compartment 113 of the device.

[0054] At least one type of dNTP (e.g., dATP, dCTP, dGTP, dTTP / dUTP) in the sample solution 114 can be modified with a redox tag 124 to form a redox-labeled dNTP 126. Non-limiting examples of redox molecules that can be used as redox tags 124 include ferrocene and its derivatives, osmium and ruthenium complexes, tetrathiafulvalene, methylene blue, anthraquinone, phenothiazine, aminophenol, nitrophenol, erythrosine B, ATTO MB2, and the like.

[0055] The redox tag 124 on the redox-labeled dNTP 126 can have similar electrochemical behavior when freely floating in the sample solution 114 as when incorporated into the amplified polynucleotide chain. For example, if the redox tag 124 is initially in its reduced form, the redox-labeled dNTP 126 can first interact with the oxidation electrode, where the redox tag 124 can be oxidized. The oxidation current passing through the electrode can then be measured. Some dNTPs with the redox tag 124 can reach the other electrode by diffusion, and then the redox tag 124 can be reduced back. This reduction reaction can induce a redox current toward the second electrode. The redox tag 124 can initially be in its oxidized form. In this case, the opposite configuration can be utilized, in which the oxidized redox tag 124 is first reduced. The current measured from the oxidized and reduced redox tag 124 can form the pre-amplification baseline.

[0056] Signaling mechanisms such as Figure 3 As shown. Nucleic acid amplification includes incorporating dNTPs into a polynucleotide chain by a polymerase enzyme. The reaction begins by combining a primer and a template (or target) polynucleotide chain. The polymerase enzyme is then incorporated into the dNTPs according to the template or target sequence to synthesize a copy of the target polynucleotide chain. Once the nucleic acid amplification reaction begins in the sample solution 114, the signal can occur simultaneously via two different pathways. Amplification can be performed in the bulk solution. Amplification can also be performed on the nanogap dielectric material 108 because the sample solution 114 contains a target polynucleotide chain 122, a polymerase 120, a primer 118, and dNTPs.

[0057] Depending on the specific requirements of the test, the amplification process may be further controlled by test conditions such as temperature, light, or pH. For example, the polymerase enzyme may be inactivated and maintained in an inactivated state. When the temperature or pH of the sample solution 114 is changed, the polymerase 120 in the sample solution 114 may be induced to become active, thereby initiating an amplification reaction. Similarly, treatment with light of a specific wavelength may induce the polymerase to become active. In this way, treating the sample solution 114 with light of a specific wavelength that can activate the polymerase 120 in the sample solution 114 may initiate an amplification reaction. Figure 3As shown, the amplification product in the bulk solution 128 can have a redox-modified base 126, and thus can contribute to the electrical signal output when the amplification product 128 diffuses into the vicinity of the active sensing region 130 or binds to the fixed primer 118b in the nanogap 108. The active sensing region 130 is defined by the space between the metal electrode 106 and the metal electrode 110 on the dielectric or nanogap 108. Alternatively, when synthesis occurs on the nanogap 108 with the fixed primer 118b and the redox-modified nucleotide 126 is incorporated into the copy, the current can also change. The induced signal change in the current indicates the presence of the target molecule, which can be manifested in the form of an increased baseline current, spikes, and fluctuations during the early stages of amplification. The time required to detect the change in the current signal change can achieve quantification of the target molecule.

[0058] The principles behind the system can be illustrated using two extreme examples: low target concentration and high target concentration. Figures 4A to 4F Detection of redox-labeled nucleotides 126 in a sample with a low concentration of target DNA 122 is shown. In this case, the probability of target DNA 122 reaching the surface-bound primer 118 in the sensing region 130 by diffusion is low. Therefore, more time may be required to detect a signal change in the current (see Figure 4A ). The time to detect the signal may be inversely proportional to the target concentration in the sample. To shorten the detection time, primers and polymerase may be added to the bulk solution 114. In this case, the target DNA 122 may begin to amplify in the bulk solution 114, and the amplified DNA with the redox label 128 may become the main population (see Figure 4C The amplified DNA with redox label 128 can eventually hybridize with the surface-bound primer 118 in the sensing region 130 (see Figure 4E ). The sensor can detect the increased current caused by the redox label within the amplified DNA. In this way, when the amplified DNA with the redox label 128 enters the sensing region 130, a significant change in current can be detected by the sensor.

[0059] like Figure 5A and 5B As shown, when the sample contains a high target DNA concentration 122, the probability of the target DNA molecules 122 reaching the surface-bound primers by diffusion is high. In this case, the redox-labeled dNTPs 126 can be incorporated during synthesis by the polymerase 120 and retained within the sensing region 130 (see Figure 5C), which can induce detectable changes in current at early time points. Overall, the simultaneous use of DNA amplification on the surface and bulk DNA amplification followed by binding to the surface can lead to faster detection of target DNA in a sample 122 regardless of the target DNA concentration, as the two methods can occur in parallel and complement each other.

[0060] Figure 6 The configuration of the electrical measurement setup is shown. The two metal electrodes can be controlled by two independent measurement electronics units, which can apply different voltages when measuring the current. For example, Figure 6 As shown, the first metal electrode 106 can apply a voltage V M1 And measure the current I M1 Similarly, the second metal electrode 110 can be applied with a voltage V M2 And measure the current I M2 A common reference electrode is used to ground the solution and provide a stable potential reference for the measurement.

[0061] Figure 7A and 7B An example of a voltage profile is described in . The voltage condition of each electrode can be selected based on the redox potential of the redox tag molecule. Figure 7A and 7B As shown, one electrode can have a voltage higher than the oxidation potential of the redox tag, and the other electrode can have a voltage lower than the redox potential of the redox tag. The voltage conditions can be further adjusted to optimize the tunneling current.

[0062] In some embodiments, the MIM electrode unit can be further processed to improve signal quality. Figure 8 As shown, the interstitial dielectric can be selectively etched to expose additional electrode surfaces and provide nanocavities. This additional step can enhance the surface reactivity of active sensing components (e.g., electrodes and dielectrics). The additional exposed electrode surfaces can also enhance the physical interaction between biomolecules and the sensor.

[0063] Figures 9-13 show examples of device designs that can be used in some embodiments. Figure 9 shows an embodiment in which the first metal electrode 106 at the bottom is not completely etched away, thereby maintaining a large surface area. As described in Figure 1, the active sensing area can be formed by etching the electrode stack in the form of a window. Therefore, the sensing area can be set along the periphery of the window. It should be appreciated that the window etched into the electrode stack can be formed of any shape. In Figure 9, a rectangular shape according to one embodiment is shown. As shown in Figure 10A and 10B As shown, for example, the shape of the window may be oval. Figure 10C and 10D A polygonal shaped window opening is shown as an example. Figure 11A and 11B An example device design with oval-shaped windows and hole compartments is shown, where the first electrode (metal electrode 1) has not been completely etched away. Figure 11C and 11D A similar example design with a polygonal shaped window opening is shown. Since the window opening can be used as a hole compartment for reagent reactions, the design may be related to the function of the opening. For example, the design of the window opening can be selected based on the effect of the shape on parameters including but not limited to filling, washing and solution flow. Depending on the properties of the surface and the composition of the sample solution, the design can be optimized to avoid problems including but not limited to bubble capture or crosstalk with adjacent holes (for example in the case of multiplexing). Figure 12 shows an embodiment in which the active sensing area is in a single-line form. Figure 13 shows another embodiment, which is designed with flattened electrodes in which the active sensing component faces upward. Since the nanogap electrodes are small, they can be easily integrated with nanochannels or microchannels to improve control of reagents and samples.

[0064] In yet another embodiment, an array of active sensing components can be formed. For example, such an array can be used to perform multiplexed testing. Figure 14 A simplified version of the array design of the sensor is shown. The array can function as a microwell array or in combination with a microfluidic or nanofluidic channel network. This format can be used to perform multiplexed testing. Multiplexed testing can include providing samples from different patients in different microwells (also referred to as well compartments) of the array so that multiple patient samples can be tested simultaneously. Multiplexed testing can also include providing primers designed to detect unique target polynucleotide chains in different microwells of the array so that more than one target polynucleotide chain can be detected using the same array. Multiplexed testing can also utilize a combination of these situations.

[0065] Figures 15 to 18 show examples of dNTPs modified with electroactive labels that can be enzymatically incorporated into a target polynucleotide chain. Figures 15A-15J An example of a dNTP modified with the electroactive labeling of ferrocene is shown. Figures 16A-16C An example of electroactive labeling of modified dNTPs with anthraquinone is shown. Figures 17A-17B An example of electroactive labeling of modified dNTPs with methylene blue is shown. Figures 18A-18C Shown is an example of a dNTP modified with a phenothiazine electroactive label. As shown, the electroactive label is covalently attached to the base of the nucleotide. In this way, the electroactive label can be retained on the polynucleotide chain.

[0066] In at least some embodiments, a system for detecting polynucleotide chains in a sample is provided. The system may include a device comprising a metal-insulator-metal (MIM) electrode unit, wherein a first metal electrode is separated from a second metal electrode by a nanogap dielectric. As described above, the unit may be formed as an electrode stack. The stack may be etched to form a pore compartment having a size suitable for receiving a sample solution. Primers for nucleic acid amplification may be fixed on the surface of a thin dielectric nanogap material. In order to amplify a target polynucleotide chain, a controller may be configured to add a sample solution containing a polymerase enzyme, dNTPs, a template, and primers to the pore compartment of the MIM electrode unit. At least one type of nucleotide may be labeled with a redox tag, which may enhance the transmission of electrons through the nanogap via a tunnel or diffusion-based redox cycle. The controller may be configured to guide the sample solution and additional reagents to flow through a microchannel or nanochannel network in fluid communication with the MIM electrode unit or with the cell array. The controller may further be configured to guide a signal (e.g., a voltage pulse) through the first and second electrodes, and measure the current value from the first electrode and the second circuit to determine a baseline signal. The controller may also be configured to measure changes in the baseline signal over a period of time to detect the presence and quantify the target polynucleotide strands.

[0067] Although exemplary embodiments have been described above, these embodiments are not intended to describe all possible forms of the present invention. On the contrary, the wording used in the specification is descriptive rather than restrictive, and it should be understood that various changes may be made without departing from the spirit and scope of the present invention. In addition, the features of the various implemented embodiments may be combined to form further embodiments of the present invention.

Claims

1. A method for detecting a polynucleotide chain in a sample, comprising: providing an apparatus comprising a nanogap sensor comprising a first electrode and a second electrode separated by a dielectric layer, wherein a primer is attached to a surface of the dielectric layer; providing a sample to the device, wherein the sample is a solution comprising a primer, a polymerase, a target polynucleotide chain, and at least one nucleotide modified with a redox tag; transmitting a first signal to the first electrode to generate a first current value; transmitting a second signal to the second electrode to generate a second current value; measuring the first current value and the second current value to determine a baseline signal; and Changes in the baseline signal are measured to detect the presence of the target polynucleotide strand. 2 . The method of claim 1 , further comprising measuring a redox current value and a tunneling current value to determine the baseline signal. 3 . The method of claim 1 , further comprising determining a differential current value between the first electrode and the second electrode to detect the presence of the target polynucleotide chain. The method according to claim 1 , further comprising inducing the incorporation of nucleotides modified with redox tags into the target polynucleotide chain by changing the pH of the sample solution. The method according to claim 1 , further comprising inducing the incorporation of nucleotides modified with a redox tag into the target polynucleotide chain by treating the sample solution with light of a wavelength capable of activating an inactive polymerase.

6. The method of claim 1, wherein one of the first signal or the second signal induces oxidation and the other of the first signal or the second signal induces reduction.

7. The method of claim 1, wherein the sample solution remains in the well compartment during the duration of amplification and detection of the target polynucleotide chain. 8 . The method of claim 1 , further comprising measuring a time between establishing a baseline signal in the sample solution and detecting a change in the baseline signal to quantify the amount of target molecules in the sample solution.

9. A method for detecting a polynucleotide chain in a sample, comprising: Providing an apparatus comprising a nanogap sensor, the nanogap sensor comprising a first electrode, a dielectric layer disposed on the first electrode, and a second electrode disposed on the dielectric layer to form an electrode stack, wherein the electrode stack comprises a well compartment sized to receive a sample solution, and wherein the dielectric layer comprises a nanocavity having a primer attached therein; providing the sample solution to the device, wherein the sample solution comprises a primer, a polymerase, a target polynucleotide chain, and at least one nucleotide modified with a redox tag; delivering a first signal to the first electrode to generate a first current value, and delivering a second signal to the second electrode to generate a second current value; measuring the first current value and the second current value to determine a baseline signal; and Changes in the baseline signal are measured to detect the presence of the target polynucleotide strand.

10. The method of claim 9, further comprising measuring a change in electron transfer between the first electrode and the second electrode, the change induced by: (i) binding of a target polynucleotide chain amplified with a redox-tagged nucleotide to a primer in the nanocavity and (ii) amplifying the target polynucleotide chain with a redox-tagged nucleotide in a bulk solution.

11. The method of claim 9, wherein the sample solution is retained in the pore compartment during the duration of amplification and detection of the target polynucleotide chain. 12 . The method of claim 9 , further comprising measuring a time between establishing the baseline signal in the sample solution and detecting a change in the baseline signal to quantify the amount of target molecules in the sample solution.

13. A system for detecting polynucleotide chains in a sample, comprising: a device comprising a nanogap sensor comprising a first electrode, a dielectric layer disposed on the first electrode, and a second electrode disposed on the dielectric layer to form an electrode stack, wherein the electrode stack comprises a well compartment sized to receive a sample solution, wherein the sample solution comprises a primer, a polymerase, a target polynucleotide chain, and at least one nucleotide modified with a redox tag, and wherein the primer is attached to the dielectric layer in fluid communication with the sample solution; and A controller configured to: When the sample solution is in the device, transmitting a first signal to the first electrode to generate a first current value, and transmitting a second signal to the second electrode to generate a second current value; measuring the first current value and the second current value to determine a baseline signal; and Changes in the baseline signal are measured to detect the presence of the target polynucleotide strand.

14. The system of claim 13, wherein the system further comprises an array of devices.

15. The system of claim 14, wherein the array of devices is configured for multiplexed testing.

16. The system of claim 13, further comprising a network of microfluidic channels in fluid communication with the nanogap sensor.

17. The system of claim 13, wherein the dielectric layer further comprises a nanocavity.

18. The system of claim 13, wherein the aperture compartment comprises an oval-shaped aperture.

19. The method of claim 13, wherein the pore compartments comprise polygonal-shaped openings. 20 . The method of claim 13 , wherein the electrode stack is partially etched to form a well compartment having the first electrode as a bottom surface of the well compartment.

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