Ferrocene-labeled primer for electrochemical detection

Abstract

The present invention provides a novel primer to which a ferrocene label is attached. The ferrocene label is incorporated into the amplification product. When the amplification product incorporating the ferrocene label is denatured, it binds to the capture probe, and the presence or absence of the ferrocene label can be detected by electrochemical detection. This system avoids the use of signal probes in sandwich assays as historically used during electrochemical detection.

Description

Technical Field

[0001] The present invention generally relates to the fields of electrochemical detection techniques and molecular diagnostics.

Background Art

[0002] Access to diagnostic tests is important to help limit the spread of disease. However, during the COVID-19 pandemic, access to effective diagnostic tests was a major problem, especially during the initial outbreak in 2020. The acceleration of COVID-19 test development and the scaling up of manufacturing and supply were important in the fight against COVID-19. There remains a need to rapidly develop accurate and rapid assays and manufacture them in order to quickly bring them to market when new variants of COVID-19 and new infectious diseases arise.

Summary of the Invention

[0003] Provided herein are methods, devices, and systems for amplifying and detecting nucleic acids, such as DNA and / or RNA, using signal primers for use in an electrochemical detection system. Amplifying and detecting nucleic acids (such as DNA and / or RNA) using signal primers accelerates the development of detection assays, improves their accuracy, shortens the time to result, and simplifies manufacturing to ensure that diagnostic kits can be rapidly developed and delivered to patients in need. The rapid development and distribution of diagnostic kits will help reduce the spread of disease.

[0004] In a first aspect, a method for detecting the presence or absence of a target nucleic acid in a sample, comprising: (a) combining a solution containing or suspected of containing the target nucleic acid with an amplification reagent to amplify the target nucleic acid if present, the amplification reagent comprising a labeled primer capable of hybridizing to the target nucleic acid, combining a solution containing or suspected of containing the target nucleic acid with an amplification reagent to amplify the target nucleic acid if present; (b) amplifying the target nucleic acid to produce a double-stranded labeled amplicon if the target nucleic acid is present; (c) incubating the double-stranded labeled amplicon with an exonuclease to form a single-stranded labeled amplicon; (d) hybridizing the single-stranded labeled amplicon to a first capture probe; and (e) detecting the presence or absence of the target nucleic acid in the sample using electrochemical detection. In some embodiments, the labeled primer, double-stranded labeled amplicon, and / or single-stranded labeled amplicon comprises at least one label selected from the group consisting of ferrocene, methylene blue, or osmium. In certain embodiments, the at least one label is ferrocene. In some embodiments, the labeled primer comprises a ferrocene label at the 5'-end, at any one of nucleotides 1-3 at the 5'-end, at the 3'-end, at any one of nucleotides 1-3 at the 3'-end, or at both the 5'-end and the 3'-end. In some embodiments, the labeled primer comprises a ferrocene label inside the labeled primer. In some embodiments, the labeled primer, double-stranded labeled amplicon, and / or single-stranded labeled amplicon comprises multiple ferrocene labels at at least two locations. In some embodiments, the single-stranded labeled amplicon comprises a first portion capable of hybridizing to the first capture probe, a second portion incapable of binding to the capture probe, and a third portion comprising at least one label. In certain embodiments, the label is selected from the group consisting of ferrocene, methylene blue, or osmium.In some embodiments, the labeled primer comprises a first portion capable of hybridizing to the first capture probe, a second portion comprising a linker, and a third portion comprising at least one label, wherein the linker connects the first portion and the third portion. In some embodiments, the method further comprises separating the labeled primer from the single-stranded labeled amplicon prior to electrochemical detection. In another embodiment, the method further comprises separating the labeled primer from the double-stranded labeled amplicon prior to electrochemical detection. In some embodiments, the labeled primer consists of a first portion capable of hybridizing to a capture probe and a second portion comprising at least a first label. In some embodiments, the amplification reagent comprises a nucleic acid polymerase, deoxynucleotide triphosphates (dNTPs), a reaction buffer necessary for the function of the nucleic acid polymerase, and a divalent cation (e.g., Mg. 2+ and / or Mn 2+ etc.). In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further comprises the step of reverse transcribing the RNA using reverse transcriptase to generate cDNA. In some embodiments, the nucleic acid polymerase is a DNA polymerase. In some embodiments, the nucleic acid polymerase further exhibits reverse transcriptase activity.

[0005] In a second aspect, a method for detecting the presence or absence of a target nucleic acid in a sample, the method comprising: (a) receiving the sample; (b) extracting nucleic acid from the sample, wherein the nucleic acid is suspected of containing the target nucleic acid; (c) combining the nucleic acid with an amplification reagent and, if present, amplifying the target nucleic acid, wherein the amplification reagent comprises a signal primer capable of hybridizing to the target nucleic acid; (d) if the target nucleic acid is present, amplifying the target nucleic acid to generate a double-stranded signal amplicon; (e) incubating the double-stranded signal amplicon with an exonuclease to form a single-stranded signal amplicon; (f) hybridizing the single-stranded signal amplicon to a first capture probe; and (g) detecting the presence or absence of the target nucleic acid in the sample using electrochemical detection. In some embodiments, the signal primer comprises an electrochemically detectable label. In some embodiments, the signal primer comprises at least one label selected from the group consisting of ferrocene, methylene blue, or osmium. In certain embodiments, the at least one label is ferrocene. As used herein, a detectable label can act as a signal transduction moiety. In some embodiments, the signal primer does not bind / hybridize to the first capture probe. In some embodiments, a first portion of the single-stranded signal amplicon binds / hybridizes to the first capture probe and a second portion of the single-stranded signal amplicon binds / hybridizes to a second capture probe. In some embodiments, the labeled primer, double-stranded labeled amplicon, and / or single-stranded labeled amplicon comprise at least one label selected from the group consisting of ferrocene, methylene blue, or osmium. In certain embodiments, the at least one label is ferrocene. In some embodiments, the labeled primer comprises a ferrocene label at the 5'-end, at any one of nucleotides 1-3 of the 5'-end, at the 3'-end, at any one of nucleotides 1-3 of the 3'-end, or at both the 5'-end and the 3'-end.In some embodiments, the labeled primer contains a ferrocene label inside the labeled primer. In some embodiments, the labeled primer, double-stranded labeled amplicon, and / or single-stranded labeled amplicon contain multiple ferrocene labels at at least two locations. In some embodiments, the single-stranded labeled amplicon includes a first portion capable of hybridizing to the first capture probe, a second portion incapable of binding to the capture probe, and a third portion containing at least one label. In certain embodiments, the label is selected from the group consisting of ferrocene, methylene blue, or osmium. In some embodiments, the labeled primer includes a first portion capable of hybridizing to the first capture probe, a second portion including a linker, and the third portion containing at least one label, wherein the linker connects the first portion and the third portion. In some embodiments, the method further includes separating the labeled primer and the single-stranded labeled amplicon prior to electrochemical detection. In another embodiment, the method further includes separating the labeled primer and the double-stranded labeled amplicon prior to electrochemical detection. In some embodiments, the labeled primer consists of a first portion capable of hybridizing to the capture probe and a second portion containing at least a first label. In some embodiments, the amplification reagents include a nucleic acid polymerase, deoxynucleotide triphosphates (dNTPs), a reaction buffer necessary for the function of the nucleic acid polymerase, and divalent cations (e.g., Mg. 2+ and / or Mn 2+ etc.). In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further includes a step of reverse transcribing the RNA using reverse transcriptase to generate cDNA. In some embodiments, the nucleic acid polymerase is a DNA polymerase. In some embodiments, the nucleic acid polymerase further exhibits reverse transcriptase activity.

[0006] In a third aspect, a process for detecting the presence of a target single-stranded or double-stranded nucleic acid in a sample, the process comprising: (a) providing (i) a sample suspected of containing the target nucleic acid, (ii) a nucleic acid primer comprising a nucleic acid sequence complementary to at least a portion of the target nucleic acid, and a first electrochemically detectable label, and (iii) a reagent for performing nucleic acid strand extension; (b) forming a reaction mixture comprising (i), (ii) and (iii) above; (c) contacting the nucleic acid primer with the target nucleic acid, if present, under hybridization conditions; (d) extending the nucleic acid primer thereby incorporating the electrochemically detectable label into the amplicon and forming an electrochemically labeled amplicon if the target nucleic acid is present; (e) denaturing the electrochemically labeled amplicon; (f) hybridizing the electrochemically labeled amplicon with a capture probe bound to an electrode surface; (g) detecting the presence of the target nucleic acid by detecting energy transfer between the electrochemically labeled amplicon and the electrode surface. In some embodiments, the electrochemically detectable label is selected from the group consisting of ferrocene, methylene blue or osmium. In certain embodiments, at least one label is ferrocene. As used herein, a detectable label can act as a signal transduction moiety. In some embodiments, the nucleic acid primer comprises a first portion that binds / hybridizes to the capture probe and a second portion that does not bind / hybridize to the capture probe. In some embodiments, the nucleic acid primer further comprises a second electrochemically detectable label, wherein the first electrochemically detectable label and the second electrochemically detectable label are different. In some embodiments, the nucleic acid primer is a signal primer that does not bind / hybridize to a first capture probe. In some embodiments, a first portion of a single-stranded signal amplicon binds / hybridizes to a first capture probe and a second portion of the single-stranded signal amplicon binds / hybridizes to a second capture probe.In some embodiments, the nucleic acid primer, double-stranded labeled amplicon, and / or single-stranded labeled amplicon comprise at least one label selected from the group consisting of ferrocene, methylene blue, or osmium. In certain embodiments, the at least one label is ferrocene. In some embodiments, the nucleic acid primer comprises a ferrocene label at the 5'-end, at any one of nucleotides 1-3 of the 5'-end nucleotide, at the 3'-end, at any one of nucleotides 1-3 of the 3'-end nucleotide, or at both the 5'-end and the 3'-end. In some embodiments, the nucleic acid primer comprises a ferrocene label inside the labeled primer. In some embodiments, the nucleic acid primer, double-stranded labeled amplicon, and / or single-stranded labeled amplicon comprise multiple ferrocene labels at at least two locations. In some embodiments, the single-stranded labeled amplicon comprises a first portion capable of hybridizing to the first capture probe, a second portion incapable of binding to the capture probe, and a third portion comprising at least one label. In certain embodiments, the label is selected from the group consisting of ferrocene, methylene blue, or osmium. In some embodiments, the nucleic acid primer comprises a first portion capable of hybridizing to the first capture probe, a second portion comprising a linker, and the third portion comprising at least one label, wherein the linker connects the first portion and the third portion. In some embodiments, the process further comprises separating the nucleic acid primer and the single-stranded labeled amplicon prior to electrochemical detection. In another embodiment, the process further comprises separating the nucleic acid primer and the double-stranded labeled amplicon prior to electrochemical detection. In some embodiments, the nucleic acid primer consists of a first portion capable of hybridizing to a capture probe and a second portion comprising at least a first label. In some embodiments, the reagents for performing nucleic acid strand extension are nucleic acid polymerase, deoxynucleotide triphosphates (dNTPs), a reaction buffer required for the function of the nucleic acid polymerase, and a divalent cation (e.g., Mg. 2+ and / or Mn 2+including (etc.). In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA and the method further includes the step of reverse transcribing the RNA using reverse transcriptase to generate cDNA. In some embodiments, the nucleic acid polymerase is a DNA polymerase. In some embodiments, the nucleic acid polymerase further exhibits reverse transcriptase activity.

Brief Description of the Drawings

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[0008] **Definitions** As used in the claims and this specification, the following terms have the following definitions.

[0009] As used herein, "amplification" refers to any in vitro method for increasing the copy number of a nucleotide sequence using a polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a nucleic acid molecule (e.g., DNA) or primer, thereby forming a new nucleic acid molecule complementary to the nucleic acid template. The nucleic acid molecule formed and its template can be used as templates for synthesizing further nucleic acid molecules. As used herein, one amplification reaction can consist of multiple nucleic acid syntheses. Amplification reactions include, for example, polymerase chain reaction (PCR). One PCR reaction can consist of 5 to 100 "cycles" of nucleic acid denaturation and synthesis.

[0010] An "amplification primer" or "primer" is a primer for amplification of a target sequence by primer extension.

[0011] An "amplicon" is a nucleic acid molecule that includes a primer or part of a primer and a newly synthesized strand that is complementary to the sequence downstream of the primer binding site. The extension product results from hybridization of the primer to a template containing the complementary sequence and extension of the primer by a polymerase using the template.

[0012] "Analyze" means to measure, detect, or determine the presence, absence, or composition of something.

[0013] An "analyte" can be anything that can selectively bind to a capture binding ligand. The analyte can be natural, biological, or synthetic, such as any of the synthetic or other molecules used in drug creation that exhibit an unusually good or specific binding affinity for the "capture binding ligand". Both the analyte and the capture binding ligand can consist of one or more different domains. One of ordinary skill in the art will understand that a complementary orientation between the analyte and the capture binding ligand is required. Suitable analytes include organic and inorganic molecules, including biomolecules. In one embodiment, the analyte is an environmental contaminant (including pesticides, insecticides, toxins, etc.); a chemical substance (including solvents, organic materials, etc.); a therapeutic molecule (including therapeutic drugs and abused drugs, antibiotics, etc.); a biomolecule (including hormones, cytokines, proteins, lipids, carbohydrates, cell membrane antigens and receptors (nerve, hormone, nutrient, and cell surface receptors) or their ligands, etc.); whole cells (including prokaryotic cells (such as pathogenic bacteria) and eukaryotic cells (including mammalian tumor cells)); viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); and spores, etc.

[0014] Suitable nucleic acid target analytes include, but are not limited to, orthomyxoviruses (e.g., influenza viruses), paramyxoviruses (e.g., respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g., rubella virus), parvoviruses, poxviruses (e.g., variola virus, vaccinia virus), enteroviruses (e.g., poliovirus, coxsackievirus), hepatitis viruses (including A, B, and C), herpesviruses (e.g., herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus), rotaviruses, norwalk virus, hantaviruses, arenaviruses, rhabdoviruses (e.g., rabies virus), retroviruses (including HIV, HTLV-I, and -II), papovaviruses (e.g., papillomavirus), polyomaviruses, and picornaviruses, as well as bacteria (Bacillus; Vibrio, such as Vibrio cholerae; Escherichia, such as enterotoxigenic Escherichia coli, Shigella, such as Shigella dysenteriae; Salmonella, such as Salmonella typhi; Mycobacterium, such as Mycobacterium tuberculosis, Mycobacterium leprae; Clostridium, such as Clostridium botulinum, Clostridium tetani, C. difficile, C. perfringens; Corynebacterium, such as Corynebacterium diphtheriae; Streptococcus, Streptococcus pyogenes, Streptococcus pneumoniae; Staphylococcus, such as Staphylococcus aureus; Haemophilus, such as Haemophilus influenzaeHaemophilus influenzae); Neisseria, e.g., Neisseria meningitidis, Neisseria gonorrhoeae; Yersinia, e.g., Yersinia pestis; Pseudomonas, e.g., Pseudomonas aeruginosa, Pseudomonas putida; Chlamydia, e.g., Chlamydia trachomatis; Bordetella, e.g., Bordetella pertussis; Treponema, e.g., Treponema pallidum, etc., including a variety of pathogenic and non-pathogenic prokaryotes for various purposes (collectively referred to as "bacterial and viral targets").

[0015] Suitable nucleic acid target analytes include, but are not limited to, nucleic acids of any number of Gram-positive organisms including Bacillus cereus group, Bacillus subtilis group, Corynebacterium, Cutibacterium acnes, Propionibacterium acnes, Enterococcus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus, Listeria, Listeria monocytogenes, Micrococcus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus, Streptococcus agalactiae (GBS), Streptococcus anginosus group, Streptococcus pneumoniae, Streptococcus pyogenes (GAS), and resistance genes mecA, mecC, vanA, or vanB (collectively "Gram-positive targets").

[0016] Suitable nucleic acid target analytes include, but are not limited to, nucleic acids of any number of Gram-negative organisms including Acinetobacter baumannii, Bacteroides fragilis, Citrobacter, Cronobacter sakazakii, Enterobacter (non-Cloacae complex), Enterobacter cloacae complex, Escherichia coli, Fusobacterium nucleatum, Fusobacterium necrophorum, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae group, Morganella morganii, Neisseria meningitidis, Proteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia, Serratia marcescens, Stenotrophomonas maltophilia, resistance genes, CTX-M, IMP, KPC, NDM, OXA (OXA-23 and OXA-48), or VIM (collectively, "Gram-negative targets").

[0017] Suitable nucleic acid target analytes include, but are not limited to, nucleic acids of any number of fungi including Candida albicans, Candida auris, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus gattii, Cryptococcus nehoformans, Fusarium or Rhodotorula (collectively "fungal targets").

[0018] In some embodiments, the target is a human-specific infectious pathogen or target, and the marker or target is a nucleic acid marker.

[0019] "Array" means a plurality of distinct sites having different capture binding ligands. In some embodiments, the array is "addressable" as long as the individual sites have a defined or determinable position relative to each other, optionally with the aid of an electronic connector and / or software.

[0020] "Capture binding ligand" is synonymous with "capture probe" or "capture binding probe" and is a compound that exhibits a relatively strong or specific affinity for another compound such that it can separate that compound from a group of other compounds in a mixture of compounds. The capture binding ligand can be a protein, carbohydrate, nucleic acid, small molecule, or any combination thereof.

[0021] The "double-stranded signal amplicon" means a double-stranded amplicon produced by the use of primers in PCR that contain an electrochemically detectable label. The double-stranded signal amplicon contains nucleic acid and signal primers.

[0022] The "electrode" means a composition that can sense current or charge when connected to an electronic device and convert it into a signal. Electrodes are known in the art and include, but are not limited to, certain metals and their oxides including gold, platinum, palladium, silicon, aluminum; metal oxide electrodes including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo206), tungsten oxide (W03), and ruthenium oxide; and carbon (including glassy carbon electrodes, graphite, carbon paste).

[0023] "Electrochemical detection" means the use of at least two electrodes to apply a potential and measure the current generated by a chemical reaction. For clarity, "electrochemical detection" excludes (i) detection of the conductivity, impedance or capacitance of a droplet, a part of a droplet, or the contents of a droplet; (ii) detection by electrochemiluminescence; and (iii) detection by optical means.

[0024] As used herein, the terms "electron-donating moiety", "electron-accepting moiety", and "electron-transfer moiety", or grammatical equivalents thereof, refer to molecules that can transfer electrons under certain conditions. It should be understood that the abilities of electron donors and acceptors are relative. That is, a molecule that can lose electrons under certain experimental conditions can accept electrons under different experimental conditions. The number of possible electron-donating and electron-accepting moieties is very large, and those skilled in the art of electron-transfer compounds can utilize several compounds, and it should be understood that the selection of those compounds is within the scope of the skill of those skilled in the art. One advantage of redox-mediated electron detection is that there are various different electron-transfer moiety labels, each having a unique distinct potential that can be selectively measured or filtered. Some electron-transfer moieties include, but are not limited to, transition metal complexes, organic electron-transfer moieties, electrodes, metallocenes such as ferrocene, and ferrocene derivatives or methylene blue or osmium.

[0025] As used herein, the terms "hybridization" and "hybridize" refer to the pairing of two complementary single-stranded nucleic acid molecules (RNA and / or DNA) that gives a double-stranded molecule. As used herein, two nucleic acid molecules can hybridize, but the base pairing is not completely complementary. Thus, mismatched bases do not prevent the hybridization of two nucleic acid molecules if appropriate conditions well known in the art are used.

[0026] The term "immobilize" or derivatives thereof includes attachment, association or binding, whether covalent or non-covalent.

[0027] "Infectious disease" means a disease characterized by an infectious disease marker, pathogen or target, whether viral, bacterial or fungal. Exemplary infectious disease targets include, for example, natural, synthetic or amplified biomolecules such as: viral targets, gram-negative targets, gram-positive targets and fungal targets.

[0028] As used herein, a "label" means something that can be signal-transmitted or stimulated to signal the presence of an event or a molecule or complex of molecules. Labels can include, for example, dyes, radioactive atoms or molecules, redox-active compounds, enzymes, enzyme substrates, nucleic acids, their derivatives, and the like. Redox-active labels can have a variety of different possibilities that can be used, similar to the presence of dyes of different colors and chemically sticky compounds.

[0029] As used herein, "monolayer" or "self-assembled monolayer" or "SAM" means a relatively ordered aggregate of molecules that have chemisorbed spontaneously on a surface, where the molecules are oriented approximately parallel to each other and approximately perpendicular to the surface. Each molecule includes a functional group that attaches to the surface and a portion that interacts with adjacent molecules in the monolayer to form a relatively regular arrangement. A "mixed" monolayer includes a heterogeneous monolayer, i.e., at least two different molecules constitute the monolayer.

[0030] As used herein, "nucleic acid" or "oligonucleotide" or grammatical equivalents thereof means at least two nucleotides covalently linked together. Nucleic acids generally contain phosphodiester bonds, but in some cases, as outlined below, can include nucleic acid analogs having alternative backbones, such as phosphoramidates. Nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids, and the nucleic acids can include any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, and the like. As used herein, the term "nucleoside" includes nucleotides as well as nucleoside and nucleotide analogs, and modified nucleosides such as amino-modified nucleosides. Further, "nucleoside" includes analog structures that do not occur in nature. Thus, for example, the individual units of peptide nucleic acids, each containing a base, are referred to herein as nucleosides.

[0031] As used herein, "nucleotide" refers to a base-sugar-phosphate combination. Nucleotides are the monomeric units of nucleic acid sequences (DNA and RNA). The term nucleotide includes mono-, di- and triphosphate forms of deoxyribonucleosides and ribonucleosides and their derivatives. The term nucleotide includes, in particular, deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, etc., or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP and ddITP. As used herein, nucleotides are unlabeled.

[0032] As used herein, "polymerase" refers to any enzyme having nucleotide polymerization activity. Examples of polymerases (including DNA polymerases and RNA polymerases) include, but are not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNA polymerase, Pyrococcus furiosus (Pflu) DNA polymerase, DEEPVENT DNA polymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl / Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME) DNA polymerase, Methanobacterium thermoautotrophicum (Mth) DNA polymerase, mycobacterium DNA polymerase (Mtb.Mlep), and mutants, variants and derivatives thereof.RNA polymerases such as T3, T5, and SP6, as well as mutants, variants, and derivatives thereof, can also be used.

[0033] As used herein, "primer" refers to a synthetic or biologically produced single-stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule. Nucleic acid amplification is often based on nucleic acid synthesis by a nucleic acid polymerase or reverse transcriptase. Many such polymerases or reverse transcriptases require the presence of a primer that can be extended to initiate such nucleic acid synthesis.

[0034] As used herein, "probe" refers to a synthetic or biologically produced nucleic acid (DNA or RNA) that contains a specific nucleotide sequence that is designed or selected to be able to hybridize specifically (i.e., preferentially) to a target nucleic acid sequence under defined stringency conditions.

[0035] A "redox-active" compound or moiety means one that can transfer, shuttle, or receive electrons from another redox-active compound. Some redox-active compounds include electrodes containing ferrocene and its derivatives, methylene blue, or osmium, and metallocenes.

[0036] "Sample solution" includes any number of things including, but not limited to, body fluids (substantially any organism's blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen, including mammalian samples including human samples); environmental samples (including, but not limited to, air, agricultural, water, and soil samples); biological warfare agent samples; research samples (i.e., in the case of nucleic acids, the sample can be the product of an amplification reaction that includes both a target and signal amplification, such as a PCR amplification reaction); purified samples, such as purified genomic DNA, RNA, protein, etc.; biological samples (bacteria, viruses, genomic DNA), etc., and as will be understood by those of skill in the art, substantially any experimental manipulation may have been performed on the sample.

[0037] "Signal primer" means a primer containing an electrochemically detectable label.

[0038] "Signal probe" means a probe molecule having some kind of label that can bind to an analyte and transmit a signal indicating the presence of the analyte. For electrochemical detection, the label is often ferrocene or a ferrocene derivative, which binds to one domain of the analyte, and another domain of the analyte binds to a capture binding ligand on the surface site of the solid support (a configuration known as a "sandwich assay").

[0039] "Single-stranded signal amplicon" means an amplicon produced by the use of a primer during PCR that contains an electrochemically detectable label. The single-stranded signal amplicon contains nucleic acid and a signal primer.

[0040] "Solid support" or "support" can be any material or matrix suitable for attaching an oligonucleotide / capture probe. Such oligonucleotides and / or capture probes can be added or attached (covalently or non-covalently) to the support by any technique or any combination of techniques well known in the art. The support may be other than an aqueous phase at room temperature and includes, for example, beads, gels, columns, column matrices, multi-titer plates, paper, membranes, printed circuit boards, or other array surfaces or supports.

[0041] The term "target nucleic acid" or "target" or grammatical equivalents herein refers to the nucleic acid sequence to be amplified or detected. These include any strand of the original nucleic acid sequence to be amplified, its complementary second strand, and copies of the original sequence produced by replication or amplification. The target sequence may sometimes also be referred to as a template for the extension of hybridized primers.

[0042] The target sequence can be part of, for example, genes, regulatory sequences, genomic DNA, cDNA, mRNA, and RNA including rRNA. It can be of any length, understanding that longer sequences are more specific. As will be understood by those skilled in the art, the complementary target sequence can take many forms. For example, it can be contained within a larger nucleic acid sequence, i.e., inter alia, all or part of a gene or mRNA, within a restriction fragment of a plasmid or genomic DNA. As will be more fully outlined below, the probe is hybridized to the target sequence to determine the presence or absence of the target sequence in the sample. The target sequence may also be composed of different target domains. For example, the first target domain of the sample target sequence may hybridize to a capture probe or a part of the capture probe, and the second target domain may hybridize to a part of a different capture probe. The target domains may be adjacent or separated. The terms "first" and "second" do not mean to assign an orientation of the sequence with respect to the 5'-3' orientation of the target sequence. For example, assuming the 5'-3' direction of the complementary target sequence, the first target domain may be located either 5' to the second domain or 3' to the second domain. A target refers to a nucleic acid molecule to which a specific primer or probe can preferentially hybridize.

[0043] As used herein, "target sequence" refers to a nucleic acid sequence within a target molecule to which a specific primer or probe can preferentially hybridize.

[0044] As used herein, the term "template" refers to a double-stranded or single-stranded molecule to be amplified, synthesized, or sequenced. In the case of a double-stranded DNA molecule, denaturation of the strands to form the first and second strands is performed to amplify, sequence, or synthesize these molecules. A primer complementary to a portion of the template is hybridized under appropriate conditions, and then a polymerase (DNA polymerase or reverse transcriptase) can synthesize a nucleic acid molecule complementary to the template or a portion thereof. The newly synthesized molecule may be the same length as or shorter than the original template. Incorporation of mismatches during synthesis or extension of the newly synthesized molecule can result in one or several mismatched base pairs. Thus, the synthesized molecule need not be exactly complementary to the template. The template can be an RNA molecule, a DNA molecule, or an RNA / DNA hybrid molecule. The newly synthesized molecule can function as a template for subsequent nucleic acid synthesis or amplification.

[0045] As used herein, recombinant DNA technology and other terms used in the fields of molecular biology and cell biology will generally be understood by those of ordinary skill in the applicable art.

[0046] General description of the device and method Devices and methods for nucleic acid amplification and detection are disclosed. The device may be equipped for an electrochemical detection method. Methods and reagents for amplification of nucleic acid sequences, and their use in the detection of nucleic acids are disclosed.

[0047] In a first embodiment, an oligonucleotide primer (i.e., a signal primer) for use in a nucleic acid amplification assay is disclosed, which comprises a primer region and an electron transfer moiety (ETM) (e.g., a ferrocene label). In some embodiments, the primer does not contain a secondary structure in solution at any temperature. In some embodiments, the primer can bind to the target nucleic acid at any temperature. In some embodiments, the oligonucleotide primer is self-annealing.

[0048] Accordingly, in a first aspect, a method for amplifying one or more nucleic acid molecules, comprising: (a) mixing a sample suspected of containing one or more templates or target nucleic acid molecules or target nucleic acids with one or more signal primers described herein and reagents necessary to perform amplification; and (b) incubating the mixture under conditions sufficient to amplify one or more nucleic acid molecules complementary to all or part of the template or target molecule. The amplified nucleic acid molecule comprises one or more signal primers described herein or a portion thereof. In one aspect, an electron transfer moiety (ETM) is incorporated at or near one or both ends of the synthetic or amplified nucleic acid molecule produced by amplification. In one aspect, the electron transfer moiety (ETM) is incorporated at or near the 5' end.

[0049] In another aspect, the method is a method of performing a nucleic acid amplification assay, comprising: (a) combining a reagent for nucleic acid amplification, a nucleic acid polymerase, a target nucleic acid or a sample suspected of containing the target nucleic acid, and a modified primer, wherein the modified primer contains a detectable label, and the detectable label is ferrocene, combining the reagent for nucleic acid amplification, the nucleic acid polymerase, the target nucleic acid or the sample suspected of containing the target nucleic acid, and the modified primer; (b) cycling the mixture of (a) to provide multiple copies of an amplicon incorporating the modified primer if the target nucleic acid is present in the sample; (c) denaturing the amplicon into a single-stranded signal amplicon; (d) exposing the single-stranded signal amplicon to a capture oligonucleotide complementary to the single-stranded signal amplicon; (e) hybridizing the hybridization region of the single-stranded signal amplicon to the capture oligonucleotide; and (f) detecting the label associated with the hybridization. In some embodiments, the detection is electrochemical detection. In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further comprises reverse transcribing the RNA using reverse transcriptase to generate cDNA. In some embodiments, the nucleic acid polymerase is a DNA polymerase.

[0050] In yet another embodiment, the method is a method of performing a nucleic acid amplification assay, comprising: (a) combining a reagent, a nucleic acid polymerase, a target nucleic acid, or a sample suspected of containing the target nucleic acid, a modified primer, and a second primer, wherein the modified primer comprises a detectable label, the detectable label is ferrocene, the second primer is complementary to a second region of the target nucleic acid, and comprises a sequence of bases having no detectable label; (b) cycling the mixture of (a) to provide a plurality of copies of a double-stranded amplicon incorporating the modified primer and the second primer when the target nucleic acid is present in the sample; (c) denaturing the amplicon incorporating the modified primer and denaturing the amplicon incorporating the second primer to single strands; (d) exposing the single-stranded signal amplicon incorporating the modified primer to a capture oligonucleotide complementary to the single-stranded signal amplicon, and exposing the second single-stranded amplicon to a capture oligonucleotide complementary to the second single-stranded amplicon; (e) hybridizing the single-stranded signal amplicon incorporating the modified primer to the capture oligonucleotide, and hybridizing the second single-stranded amplicon to the capture oligonucleotide; and (f) detecting the label associated with the first primer incorporated into the amplicon. In some embodiments, the detection is electrochemical detection. In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further comprises reverse transcribing the RNA using reverse transcriptase to generate cDNA. In some embodiments, the nucleic acid polymerase is a DNA polymerase.

[0051] In yet another aspect, the method is a method of performing a nucleic acid amplification assay, comprising: (a) combining a reagent, a polymerase, a target nucleic acid or a sample suspected of containing the target nucleic acid, a first modified primer and a second modified primer, wherein the first modified primer contains a first detectable label, the second modified primer contains a second detectable label, the first and second detectable labels are ferrocene, and the second modified primer contains a sequence of bases complementary to a second region of the target nucleic acid; (b) cycling the mixture of (a) to provide multiple copies of a double-stranded amplicon incorporating the first modified primer and the second modified primer when the target nucleic acid is present in the sample; (c) denaturing the amplicon incorporating the first modified primer and denaturing the amplicon incorporating the second modified primer to single strands; (d) exposing the single-stranded signal amplicon incorporating the first modified primer to a capture oligonucleotide complementary to the first single-stranded signal amplicon and exposing the second single-stranded signal amplicon to a capture oligonucleotide complementary to the second single-stranded signal amplicon; (e) hybridizing the first single-stranded signal amplicon incorporating the first modified primer to the first capture oligonucleotide and hybridizing the second single-stranded signal amplicon to the second capture oligonucleotide; (f) detecting the label associated with the first primer incorporated into the amplicon and detecting the label associated with the second primer incorporated into the amplicon. In some embodiments, the detection is electrochemical detection. In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA and the method further comprises reverse transcribing the RNA using reverse transcriptase to generate cDNA. In some embodiments, the polymerase is a DNA polymerase.

[0052] Overview of the system Current electrochemical detection assays are based on the principle of nucleic acid hybridization using a sandwich assay format, where single-stranded amplicons bind to sequence-specific signal probes, and the signal probe / single-stranded amplicon complex (the "signal probe complex") binds to electrode-bound capture probes as shown in FIG. 1a, which is a schematic diagram showing signal generation using current capture probe / signal probe sandwich technology.

[0053] In contrast, the present specification provides methods and systems that eliminate the need for signal probes and signal probe complexes. The disclosed methods and systems are useful in genotyping, subtyping, isotyping or expression assays.

[0054] First, target nucleic acid is extracted from a sample and amplified using a signal primer by polymerase chain reaction (PCR) to form double-stranded signal DNA. In some embodiments, the target nucleic acid is DNA that is directly amplified using a primer pair that includes at least one signal primer. In some embodiments, the target nucleic acid can be RNA, and the RNA is subjected to reverse transcription amplification prior to amplification using polymerase chain reaction (PCR) with a signal primer to form double-stranded signal DNA. Here, the reverse transcription primer can be an additional reverse transcription primer or a signal primer. In some cases, the reverse transcription and amplification reactions can be performed in a one-step reaction (RT-amplification). The resulting double-stranded signal DNA is then digested with exonuclease to produce single-stranded signal DNA. Binding of the single-stranded signal DNA to the capture probe brings an electron transfer moiety ETM (e.g., ferrocene label) near the surface of the gold electrode. An electrical signal specific to the hybridized complex is generated as a byproduct of a redox reaction when a voltage is applied to the system (Figure 1b). Sequential analysis of each electrode enables detection of multiple analyte targets. The detection device measures and interprets this electrical output (nanoampere, nA) (e.g., in a qualitative manner such as "detected" or "not detected") to determine the result for each target nucleic acid.

[0055] In this specification, at least one "signal primer" provided herein is used for the amplification of a target nucleic acid. In some embodiments, the signal primer is designed to have a ferrocene label (such as N6, QW56, or QW80, etc.) attached to the 5'-end or near the 5'-end of a non-phosphorylated oligonucleotide primer (for example, attached to any one of nucleotides 1 to 3 at the 5'-end, or attached to any one of nucleotides 1 to 2 at the 5'-end). In some embodiments, the amplicon generated by using the signal primer has a ferrocene label at its 5'-end or near its 5'-end (for example, within nucleotides 1 to 3 or nucleotides 1 to 2 at its 5'-end). As shown in Figure 1b, the single-stranded signal amplicon binds to the capture probe, and when a voltage is applied, the ferrocene label is transported near the surface of the gold electrode to generate a signal. Therefore, the signal probe is no longer required for detection.

[0056] Prior to the present disclosure, it was unclear whether nucleic acid amplification could be performed using a primer linked to a ferrocene label. For example, it was not known whether a polymerase could recognize a primer with an ETM attached (such as a ferrocene label, etc.). It was also not known whether the ETM (such as a ferrocene label, etc.) attached to the primer could withstand thermal cycling. For example, it was not known whether the ETM (such as a ferrocene label, etc.) itself could withstand thermal cycling. It was also not known whether Taq polymerase or reverse transcriptase was inhibited by the ETM (such as a ferrocene label, etc.). Furthermore, prior to the present disclosure, it was unclear whether signal detection could occur using a primer linked to an ETM (such as a ferrocene label, etc.) in terms of whether a detectable label could be held close enough to the electrode to detect a signal.

[0057] For example, in a conventional sandwich assay of the prior art, an ETM (such as a ferrocene label or the like) is attached to a signal probe, and the signal probe binds to an amplicon. Here, the gap between the signal probe binding site and the capture probe binding site is usually 0 bases. Alternatively, the sequence gap between the signal probe and the capture probe may be 0 to 2 bases. Further, in a conventional sandwich assay system, it is known that when the ETM (such as a ferrocene label or the like) is more than 5 bases away from the capture probe, the signal decreases dramatically.

[0058] When a ferrocene label is attached to the 5'-end or near the 5'-end of an amplicon (e.g., within nucleotides 1-3 or nucleotides 1-2 of its 5'-end), as in some embodiments of the present disclosure, it is further away from the capture probe (in some cases even 100 base pairs away), and thus, one of ordinary skill in the art would not know whether such a "far-away" ferrocene label can generate a detectable signal. Accordingly, herein, a capture probe signal primer complex is provided in which an ETM (e.g., a ferrocene label or the like) is more than 5 base pairs away from the position where the capture probe hybridizes. In some embodiments, a capture probe signal primer complex is provided in which an ETM (e.g., a ferrocene label or the like) is more than 10 base pairs away from the position where the capture probe hybridizes. In further embodiments, herein, a capture probe signal primer complex is provided in which an ETM (e.g., a ferrocene label or the like) is more than 50 base pairs away from the position where the capture probe hybridizes. In further embodiments, herein, a capture probe signal primer complex is provided in which an ETM (e.g., a ferrocene label or the like) is more than 100 base pairs away from the position where the capture probe hybridizes. In further embodiments, herein, a capture probe signal primer complex is provided in which an ETM (e.g., a ferrocene label or the like) is more than 200 base pairs away from the position where the capture probe hybridizes. In some embodiments, a capture probe signal primer complex is provided herein in which an ETM (e.g., a ferrocene label or the like) is 5-100 base pairs away from the position where the capture probe hybridizes. In additional embodiments, a capture probe signal primer complex is provided herein in which an ETM (e.g., a ferrocene label or the like) is 5-500 base pairs away from the position where the capture probe hybridizes. In certain embodiments, a capture probe signal primer complex is provided herein in which an ETM (e.g., a ferrocene label or the like) is 5-50 base pairs away from the position where the capture probe hybridizes.

[0059] Electrochemical detection system The present specification further provides an electrochemical detection system for specifically detecting a target nucleic acid segment. This system utilizes a capture probe, such as a nucleic acid or peptide nucleic acid probe, which is complementary to and specifically hybridizes with the target nucleic acid segment bound to a detectable label to generate a measurable current when an amperometric potential is applied. In some embodiments, the electrochemical signal is proportional to the amount of target nucleic acid in the sample.

[0060] Compositions and methods useful for detecting a target analyte in a sample are also provided. Signal primers, single-stranded signal amplicons, and hybridization complexes bound to single-stranded signal amplicons provide new and useful methods for detecting a target analyte. Here, the signal primer used for amplification of the target nucleic acid includes a nucleic acid capable of specifically hybridizing with the target nucleic acid and an electrochemically detectable signal transduction moiety. Thus, after amplification using the signal primer, the single-stranded signal amplicon includes a nucleic acid and an electrochemically detectable signal transduction moiety. The assay complex includes a capture binding ligand bound to an electrode (e.g., a gold electrode) and a single-stranded signal amplicon attached to the capture binding ligand, and the single-stranded signal amplicon includes a nucleic acid and an electrochemically detectable signal transduction moiety.

[0061] Signal primers simplify assay development An electrochemical detection system using signal primers is described herein. Conventional electrochemical detection systems utilize a sandwich assay for detection. Thus, the amplicon must be long enough to bind to both the capture probe and the signal probe. In contrast, using the signal primers disclosed herein eliminates the need to bind a signal probe, reducing the length of the amplicon. This makes primer design easier because (1) shorter regions are required to distinguish target nucleic acids and (2) only three conserved regions are required for signal primers as disclosed herein (forward primer, reverse primer, and capture probe), as opposed to four conserved regions required for conventional sandwich assays (forward primer, reverse primer, signal probe, and capture probe). Furthermore, using an electrochemical detection moiety bound to the nucleic acid amplicon eliminates the need to reduce background signal or enhance the signal-to-noise ratio. Specifically, there is no need to include a donor / quencher dye pair, i.e., a quencher for a fluorescent donor dye or donor fluorophore.

[0062] Signal primers improve sensitivity Improved sensitivity has been observed using signal primers. Without being bound to a particular theory, in prior art sandwich assays, if the amplicon does not bind to the signal probe with 100% efficiency, the sensitivity of the assay is negatively affected. By eliminating the need for the amplicon to bind to the signal probe, the sensitivity of the assay can be improved. Inclusion of an ETM (e.g., ferrocene labeling, etc.) is thought to not interfere with amplification but may reduce the ability of the signal primer to misprime to the target nucleic acid molecule. The increase in sensitivity is particularly important for the amplification of template nucleic acids that are difficult to amplify and produce only small amounts or no desired amplification products in a PCR reaction.

[0063] Signal primers shorten the resulting time Recent studies have shown that patients with severe sepsis or septic shock showed an increased likelihood of death by 7.6% for every hour without the application of antibiotic treatment. Liang et al., Empiric Antimicrobial Therapy in Severe Sepsis and Septic Shock: Optimizing Pathogen Clearance, Curr Infect Dis Rep. 2015 Jul;17(7):493. Survival rates may increase if the detection system is performed more rapidly.

[0064] As described above, when using a signal primer for amplification, the amplicon does not require a signal probe binding region, and as a result, the amplicon can be made shorter. A shorter amplicon means that the time required for amplification is shortened. Shortening the amplification time shortens the time until the result is obtained, and thus can save the patient's life.

[0065] Furthermore, in a prior art sandwich assay, the amplicon must first bind to the signal probe during the hybridization reaction, and then the signal probe - amplicon complex binds to the capture probe. The use of a signal primer eliminates the need for a separate signal probe / amplicon hybridization step, further shortening the resulting time.

[0066] The signal primer simplifies the manufacturing process By eliminating the need for a signal probe, the detection hybridization complex is simplified from three components to two, and as a result, the manufacturing process of the diagnostic test / kits is simplified.

[0067] Differences from the prior art Certain types of signal primers are known in the art (see, e.g., U.S. Patent Nos. 8,323,929, 9,068,948, and U.S. Patent Publication No. 20190024167). However, all of these prior art signal primers utilize fluorescent labels. In contrast, the signal transduction primers provided herein use labels that can be detected by electrochemical detection. This difference is important because prior to the present disclosure, it was unknown whether nucleic acids (e.g., DNA) could be amplified in the presence of an ETM (e.g., ferrocene label, etc.). For example, it was not known whether Taq polymerase or reverse transcriptase was inhibited by an ETM (e.g., ferrocene label, etc.). More importantly, it was not known whether single-stranded signal amplicons could be detected when bound to a capture probe. This is due to the distance of the ETM (e.g., ferrocene label, etc.) from the capture probe. In conventional sandwich assay systems, the signal decreases dramatically as the signal probe moves away from the capture probe.

[0068] This embodiment does not use a reporter probe, and its 3' end hybridizes to the complement of the 5' adapter sequence of the signal primer to provide a 5' overhang as disclosed in U.S. Patent No. 8,323,929. Further, this embodiment also does not use polymerase to fill in the overhang and does not synthesize the complement of the 5' overhang of the reporter probe. This embodiment does not detect the synthesis of the reporter probe complement either directly or indirectly as an indicator of the presence of the target nucleic acid.

[0069] Furthermore, as disclosed in U.S. Patent No. 9,068,948, this embodiment does not detect the presence or amount of a target nucleic acid by detecting energy transfer between a first energy transfer element in a nucleic acid primer and a second energy transfer element in an incorporated labeled nucleotide. This embodiment does not detect energy transfer between labeled primers, between a labeled primer and a nucleotide(s), between labeled nucleotides, between a fluorescent intercalator and a labeled primer(s), or between a labeled probe and a nucleotide(s). This embodiment does not depend on the proximity between primers or nucleic acid constructs on each strand, as in U.S. Patent No. 9,068,948.

[0070] Furthermore, the signal primer of this embodiment is not immobilized as in U.S. Patent Application Publication No. 20190024167. In this embodiment, as in U.S. Patent Application Publication No. 20190024167, there is no need to reduce or quench the energy or other detectable properties released from the label.

[0071] This system does not enable real-time detection. Unlike fluorescence-based systems where the label can be monitored "in real time" while the reaction is occurring, the disclosed signal primer must bind to a capture probe for detection at the end of amplification. This is a so-called "endpoint" detection system. Therefore, the system cannot be used to provide semi-quantitative or quantitative information regarding the initial amount of target present. Nevertheless, signal generation is specific and is expected to be proportional to the presence of the appropriate target molecule in the sample. Furthermore, the signal primers provided herein require hybridization with a capture probe for detection and cannot be detected directly from the PCR product.

[0072] General Background Regarding the Value of Nucleic Acid Testing Nucleic acid tests have a wide range of uses. Most current commercial tests are for infectious diseases, including Chlamydia, gonorrhea, hepatitis, and human immunodeficiency virus (HIV) viral load; genetic diseases, including cystic fibrosis; coagulation and hematological factors, including hemochromatosis; and cancers, including breast cancer genes. Other areas of interest include cardiovascular diseases and drug resistance screening, known as pharmacogenomics. Most tests are currently performed in centralized laboratories that process hundreds, if not thousands, of samples per day.

[0073] Sequence-specific hybridization of labeled oligonucleotide probes has long been used as a means for detecting and identifying selected nucleotide sequences. Conventional detection methods for the final step of nucleic acid analysis are well known in the art and include sandwich capture methods based on radioactivity, colorimetry, fluorescence, fluorescence resonance energy transfer (FRET), and electrochemistry.

[0074] Labeling probes with ferrocene provides a relatively sensitive means for facilitating the detection of probe hybridization. Several patents deal with the electrochemical detection of nucleic acids. For example, U.S. Patent No. 10,001,476 (incorporated herein by reference in its entirety) discloses capture probe control and detection by various capture and signal probe configurations and combinations of configurations that can facilitate accurate and efficient multiplex analyte detection. U.S. Patent No. 10,001,476 discloses single-stranded DNA on an electrode (capture probe) that binds to a target. Such systems require the ferrocene label to be held in close proximity to the electrode for the ferrocene label to function. Sandwich assays have been used in the prior art to achieve proper orientation of the ferrocene label with respect to the electrode. As described above, sandwich assays require an amplicon that can bind to both the capture probe and the signal probe. The development of primers for amplifying a unique and conserved sequence that can bind to both the capture probe and the signal probe is complex. Therefore, there is a need to develop a simpler system.

[0075] Electrochemical detection The methods and systems provided herein include a single-stranded signal amplicon specific to a nucleic acid capture probe being held in proximity to a detection electrode and forming a hybridization complex on the detection electrode under conditions where it can be detected. Thus, the methods provided herein include testing for the presence of a hybridization complex on an electrode under conditions where a single-stranded signal amplicon bound to a capture probe is in proximity to the electrode and transferring electrons thereto. Single-stranded signal amplicons and signal primers not bound to the capture probe are driven away from the electrode when the voltage / current is turned on and not detected.

[0076] In some embodiments, the detection method is implemented with a single-use cartridge that uses electrochemical detection. In some embodiments, the electrochemical detection includes an electrode that includes a monolayer that includes a conductive oligomer and a capture binding ligand.

[0077] In some embodiments, the method can utilize one or more capture probes on the same detection electrode that bind to different portions of a single-stranded signal amplicon. When multiple capture probes are used, each is specific to a different portion of a common nucleic acid sequence of interest. The nucleic acid sequence of interest can be amplified, for example, by PCR. In some embodiments, the electrode has a self-assembled monolayer (''SAM''). In some embodiments, the electrode has a mixed SAM of two or more species, each species characterized by a different chain length, number of conjugation bonds (if present), and / or substituents (if present).

[0078] In some embodiments, the ETM is a detectable label such as a ferrocene label. In some embodiments, the ferrocene label is selected from the group consisting of N6, QW56, and QW80. In certain embodiments, the detectable label is N6 (see, e.g., FIG. 7). In certain embodiments, the detectable label is QW56 (see, e.g., FIG. 8). In certain embodiments, the detectable label is QW80 (see, e.g., FIG. 9). Here, the detection method is electrochemical.

[0079] In certain embodiments, the capture oligonucleotide is immobilized on a gold surface. In other embodiments, the capture oligonucleotide is immobilized on an electrode. In some embodiments, a single-stranded signal amplicon comprising a modified signal primer hybridizes to the capture oligonucleotide. In some embodiments, the label used is electron transfer moieties (ETM), and the addressable solid support detection site is the detection electrode on which the capture probe is spotted. In some embodiments, for example, in an electrochemical detection embodiment of a nucleic acid analyte using a detection electrode, the site is also characterized by an insulating self-assembled monolayer or mixed monomolecular film layer.

[0080] Electrochemical detection is known to those skilled in the art. Generally, at least a first input signal is applied to the assay complex and an output signal is received. The output signal is then processed to detect the presence of the target analyte. Some embodiments utilize a plurality of assay complexes each attached to different cells or pads of an array.

[0081] Hybridization In the above PCR reaction, the amplicon is generated by two amplification primers that generate two different single-stranded regions. After amplification, each amplified single-stranded signal amplicon can bind to the capture probe and generate a signal.

[0082] Electron transfer In some embodiments, the detection of ETM is based on electron transfer through the stacked π-orbitals of double-stranded nucleic acids. This basic mechanism is described in U.S. Patent Nos. 5,591,578, 5,770,369, 5,705,348, and PCT US97 / 20014, all of which are incorporated herein by reference. Briefly, previous studies have shown that electron transfer can proceed rapidly through the stacked π-orbitals of double-stranded nucleic acids and significantly more slowly through single-stranded nucleic acids. Thus, this can serve as the basis for an assay. Accordingly, by adding ETM to nucleic acids attached to a detection electrode via a conductive oligomer, electron transfer between the ETM and the electrode through the nucleic acid and the conductive oligomer can be detected.

[0083] Detection electrode In one embodiment, the detection electrode is formed on a substrate typically formed from a gold electrode. However, as will be appreciated by those skilled in the art, other electrodes can be used as well. The substrate can include a wide variety of materials, as will be appreciated by those skilled in the art. In one embodiment, the substrate includes a printed circuit board (PCB). Thus, generally, suitable substrates include, but are not limited to, glass fiber, Teflon, ceramic, glass, silicon, mica, plastics (including acrylic, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethane, Teflon™, and derivatives thereof, etc.), GETEK (a blend of polypropylene oxide and glass fiber), and the like.

[0084] Generally, the material includes a printed circuit board material. The circuit board material includes an insulating substrate that is coated with a conductive layer and processed using lithography techniques, particularly photolithography techniques, to form patterns of electrodes and interconnects (which may also be referred to in the art as interconnects or leads). The insulating substrate is generally a polymer, but does not necessarily have to be a polymer. As is known in the art, one or more layers can be used to fabricate either a "two-dimensional" (e.g., all electrodes and interconnects in a plane) or a "three-dimensional" (the electrodes are on one surface and the interconnects can go to the other side through the substrate) substrate. Three-dimensional systems often rely on the use of vias or etching, followed by electroplating with a metal such as copper, so that "through board" interconnects are made. The circuit board material often has a foil already attached to a substrate such as a copper foil, and additional copper, for example by electroplating, can be added as needed (e.g., for interconnects). The copper surface may then need to be roughened, for example by etching, to enable the attachment of an adhesive layer.

[0085] Accordingly, in some embodiments, a biochip (which may also be referred to herein as a "chip") is disclosed that includes a substrate that includes a plurality of electrodes such as gold electrodes. The electrodes form an array. Each electrode can include a self-assembled monolayer as outlined herein and known to those skilled in the art. In one embodiment, one of the monolayer-forming species includes a capture ligand as outlined herein and known to those skilled in the art. Further, each electrode has an interconnect that is attached to the electrode at one end and ultimately to a device that can control the electrode. That is, each electrode is independently addressable.

[0086] The substrate can be part of a larger device that includes a detection chamber that exposes a given volume of sample to the detection electrodes. Generally, the detection chamber ranges from about 1 μl to 1 ml, or from about 10 μl to 500 μl. As will be understood by those skilled in the art, smaller or larger volumes can be used depending on the experimental conditions and the assay.

[0087] In some embodiments, the detection chamber and electrodes are part of a cartridge that can be placed in a device that includes electronic components (AC / DC voltage source, ammeter, processor, readout display, temperature controller, light source, etc.). In this embodiment, the interconnections from each electrode are arranged such that when the cartridge is inserted into the device, a connection between the electrodes and the electronic components is established.

[0088] Detection electrodes on a circuit board material (or other substrate) are generally fabricated in a wide variety of ways. Generally, high-purity gold is used and can be deposited on the surface by physical vapor deposition methods (sputtering and evaporation) or solution deposition methods (electroplating or electroless processes). When electroplating is performed, the substrate must initially contain a conductive material. Glass fiber circuit boards often have a copper foil provided. In many cases, an adhesion layer between the substrate and the gold is used to ensure good mechanical stability, depending on the substrate. Thus, some embodiments utilize a deposited layer of an adhesion metal such as chromium, titanium, titanium / tungsten, tantalum, nickel, or palladium that can be deposited on the gold as described above. When electroplated metal (either the adhesion metal or the electrode metal) is used, a fine-grained additive often referred to in the industry as a brightener can optionally be added to alter the surface deposition characteristics. Brighteners are mixtures of organic and inorganic species such as cobalt and nickel.

[0089] Generally, the adhesive layer has a thickness of from about 100 Å to about 25 microns (1000 microinches). If the adhesive metal is electrochemically active, the electrode metal must be coated with a thickness that prevents "bleed-through", and if the adhesive metal is not electrochemically active, the electrode metal may be thinner. Generally, the electrode metal (gold) is deposited with a thickness in the range of from about 500 Å to about 5 microns (200 microinches), from about 30 microinches to about 50 microinches. Generally, gold is deposited to create electrodes in the size range of from about 5 microns to about 5 mm in diameter or from about 100 to 250 microns. The detection electrodes thus formed are then washed and a SAM is added as discussed below.

[0090] Accordingly, a method of fabricating a substrate including a plurality of gold electrodes is provided. The method includes first coating a substrate with an adhesive metal such as nickel or palladium (optionally including a brightener). The electrode metal such as gold is then coated over the adhesive metal via electroplating. Next, a pattern of the device including the electrodes and their associated interconnects is fabricated using lithography techniques, particularly photolithography techniques known in the art, and wet chemical etching. In many cases, a non-conductive chemical resistant insulating material such as a solder mask or plastic is laid down using these photolithography techniques, leaving only the connections to the electrodes and the exposed leads. The leads themselves are generally coated.

[0091] Self-assembled monolayer This method can continue with the addition of a SAM (not shown in FIGS. 1a and 1b) that, in some embodiments, also adheres to the electrode surface and helps prevent or reduce unwanted electron transfer events to the electrode surface, via one or more linkers in a similar format to the capture probe linker (element 2a in FIGS. 1a and 1b). In some embodiments, droplet deposition techniques are used to add the necessary species, i.e., monolayer-forming species, one of which is the capture ligand. Droplet deposition techniques are well known for creating “spot” arrays. This is done to add different compositions to each electrode, i.e., to create an array containing different capture ligands. Alternatively, the SAM species may be the same for each electrode, which may be achieved using droplet deposition techniques or immersion of the entire substrate or the surface of the substrate into a solution.

[0092] This system finds particular utility in array format, i.e., when there is a matrix of addressable detection electrodes (generally referred to herein as “pads,” “addresses,” or “micro-locations”). As used herein, an “array” means a plurality of capture ligands in array format, and the size of the array depends on the composition of the array and the end use. Arrays can be made containing from about 2 to thousands of different capture ligands. Generally, an array can contain from 2 to over 100,000, depending on the size of the electrodes and the end use of the array. Ranges are from about 2 to about 10,000, from about 5 to about 1000, and from about 10 to about 100. In some embodiments, the composition may not be in array format, i.e., in some embodiments, a composition containing a single capture ligand can be made as well. Further, in some arrays, multiple substrates may be used, either with different compositions or the same composition. Thus, for example, a large array may comprise a plurality of smaller substrates.

[0093] In one embodiment, although this is not necessary in many systems, the electrode includes a self-assembled monolayer (SAM). As outlined herein, the efficiency of target anatase binding (e.g., oligonucleotide hybridization) can increase when the analyte is away from the electrode. Similarly, the non-specific binding of biomolecules containing the target analyte to the electrode generally decreases when a monolayer is present. Thus, the monolayer facilitates the maintenance of the analyte away from the electrode surface. Further, the monolayer serves to keep charged species away from the surface of the electrode. Thus, this layer helps prevent electrical contact between the electrode and the ETM or between the electrode and charged species in the solvent. Such contact can result in a direct or indirect "short circuit" through charged species that may be present in the sample. Thus, the monolayer is densely packed into a uniform layer on the electrode surface with minimal "holes" present. Thus, the monolayer functions as a physical barrier to prevent access of the solvent to the electrode.

[0094] The SAM may contain only conductive oligomers or may contain a mixture of conductive oligomers and insulators. As outlined herein, the use of the monolayer reduces the amount of non-specific binding of biomolecules to the surface and, in the case of nucleic acids, increases the efficiency of oligonucleotide hybridization as a result of the distance of the oligonucleotide from the electrode. Thus, the monolayer facilitates the maintenance of the target analyte away from the electrode surface. Further, the monolayer serves to keep charge carriers away from the surface of the electrode. Thus, this layer helps prevent electrical contact between the electrode and the electron transfer moiety (ETM; redox active) or between the electrode and charged species in the solvent. Such contact can result in a direct or indirect "short circuit" through charged species that may be present in the sample. Thus, the monolayer is densely packed into a uniform layer on the electrode surface with minimal "holes" present. Thus, the monolayer can function as a physical barrier to block access to unwanted signals ("noise") to the solvent and the electrode.

[0095] Nucleic acid testing using a single-use device An exemplary process will be generally described. Although the various elements (steps) are described as consecutive steps having a predetermined order, it should be understood that the process is exemplary and not intended to be limiting. One of ordinary skill in the art will recognize that many of the various elements (steps) can be performed in an order different from that described herein, can be performed simultaneously or substantially simultaneously with other elements (steps), or can be completely omitted. Accordingly, the order of the described elements (steps) is not limiting.

[0096] Step 1: Load the sample. Step 2: Extract nucleic acid, such as DNA. Step 3: Combine the nucleic acid, such as DNA, with an amplification reagent containing an ETM-labeled primer (i.e., a signal primer). Step 4: Amplify the nucleic acid, such as DNA, to produce a double-stranded signal amplicon. Step 5: Incubate the double-stranded signal amplicon with exonuclease to form a single-stranded signal amplicon. Step 6: Combine the single-stranded signal amplicon with a capture probe. Step 7: Electrical sensor detection.

[0097] In some embodiments, the process of step 2 may include extracting RNA and may further include step 3a of reverse transcribing the RNA to provide cDNA.

[0098] PCR in General Polymerase chain reaction (PCR) is a relatively simple technique for amplifying a DNA template to generate a specific DNA fragment in vitro. Typical amplification reactions include target DNA, a heat-stable polymerase (e.g., DNA polymerase, etc.), two oligonucleotide primers (5' and 3'), deoxynucleotide triphosphates (dNTPs), a reaction buffer, and a divalent cation (e.g., Mg 2+ and / or Mn 2+Examples include (...). Each cycle of PCR includes steps for template denaturation, primer annealing, and primer extension. The first step is to denature the target DNA by heating. In the denaturation process, the two intertwined DNA strands separate from each other, producing single-stranded DNA templates necessary for replication by heat-stable DNA polymerase. In the next step of the cycle, the oligonucleotide primer forms a stable association (anneals) with the denatured target DNA, and the temperature is lowered so that it can function as a primer for DNA polymerase. Finally, the synthesis of new DNA begins. An enzyme called "Taq polymerase" uses the original strand as a template to synthesize ( "construct") two new strands of DNA. This process results in the replication of the original DNA, with each of the new molecules containing one old strand and one new strand of DNA. Then, each of these strands can be used to create two new copies, and so on. The cycles of denaturing and synthesizing new DNA are repeated 30 or 40 times, resulting in over a billion exact copies of the original DNA segment. The cycling process of PCR is typically automated in a thermocycler programmed to change the temperature of the reaction to enable DNA denaturation and synthesis.

[0099] There are several nucleic acid amplification techniques that differ from conventional PCR reactions in that the reaction is performed at a single temperature. These isothermal methods include cycling probe reactions, strand displacement, Invader (trademark), SNPase, rolling circle reactions, and NASBA. The methods and systems described herein can also be used with isothermal methods.

[0100] In some embodiments, in the absence of nucleic acid synthesis, the primer should not be able to bind to the capture probe, and there should be little or no electron transfer between the signal primer and the electrode. In other embodiments, in the absence of nucleic acid synthesis, the primer can bind to the capture probe, but the electron transfer is undetectable or below a predetermined limit.

[0101] The selection of appropriate PCR conditions is within the understanding of those skilled in the art. Those skilled in the art will understand that it may be necessary to adjust the temperature concentrations of the nucleic acid target, primers, and various steps to optimize the PCR reaction for a given target and primers. Such optimization does not require excessive experimentation.

[0102] Types of PCR The signal primers disclosed herein can be used in any amplification reaction, including PCR, 5-RACE, anchor PCR, "one-sided PCR", LCR, NASBA, SDA, RT-PCR, real-time PCR, quantitative PCR, quantitative RT-PCR, and other amplification systems known in the art, including the universal primer format.

[0103] In some embodiments, the signal primer has two regions: one that can bind to a DNA template during PCR amplification as described in item "i" of FIG. 2a, and one that can generate a signal during redox-mediated electron detection as described in item "ii" of FIG. 2a. In some embodiments, the signal primer has three regions, namely one region (item "i" of FIG. 2l) that can bind to a DNA template during PCR amplification, a second region (item "ii" of FIG. 2I) that can generate a signal during redox-mediated electron detection, and a third region (a linker symbolized as a triangle in FIG. 2l) that links the first and second regions.

[0104] The sequence amplified by the signal primer is selected based on a known target sequence such that amplification proceeds at a reasonably detectable rate when hybridization to a complementary target sequence occurs.

[0105] The PCR reaction can involve rapid temperature fluctuations. Here, it was not known whether signal primers could withstand these temperature fluctuations. For example, the high temperatures (e.g., 90 - 99 °C) used to denature double-stranded DNA, the low temperatures (e.g., 40 - 60 °C) at which DNA primers hybridize to the DNA template, and the hybridization temperatures (e.g., 60 - 80 °C).

[0106] Assay Development When developing an assay that utilizes a prior art sandwich assay (a capture probe bound to an amplicon bound to a signal probe), it can be difficult to find a portion of the target organism sequence that is highly conserved (i.e., detectable across many variants) and uniquely identifies the target organism. This is further complicated if the highly conserved but unique identifier must be of sufficient length to enable both capture and signal probe binding.

[0107] An important advantage of the provided methods and systems is that there is no need to develop signal probes. Put another way, there is no need to develop amplicons with signal probe binding regions. Thus, the resulting amplicons can be made much shorter, simplifying and speeding up assay design. This should result in shorter extension times, sharper melting points, and overall higher efficiency in each amplification round since the synthesis amount is reduced compared to conventional sandwich assay systems. This can also result in a higher detection signal and thus higher sensitivity.

[0108] Assays that do not utilize signal probes are described. Assays that do not utilize sandwich assays during detection are described.

[0109] When a voltage is applied, the unbound signal primer is driven away from the electrode, so there is no need to separate the signal primer from the single-stranded signal amplicon. Unlike fluorescence-based systems that require the use of a quencher, this system does not require the use of such donor / quencher dye pairs.

[0110] Design of Synthetic Oligonucleotides Regarding the design of synthetic oligonucleotides for use in amplification reactions, Rychlik et al., (1989, Nucleic Acids Research, vol 17(21):8543-8551) and Rychlik (1995, Molecular Biotechnology, vol 3:129-134) have described selection criteria and computer programs for designing probes and primers, including primers for in vitro amplification of DNA. Both teach that primers should not form secondary structures or exhibit self-hybridization. U.S. Patent No. 6,495,323 (incorporated herein by reference in its entirety) details the formation of probes attached to an electron transfer moiety. The same process used to make ferrocene-labeled probes can be used to make ferrocene-labeled primers.

[0111] Probe Synthesis, Functionalization, and Conjugation - General U.S. Patent No. 10,001,476 discloses probe synthesis, functionalization, and conjugation in more detail, the disclosure of which is incorporated herein by reference in its entirety. Probe synthesis, functionalization, and conjugation are all techniques well known in the art.

[0112] Nucleic acid capture probes are typically designed to be complementary to a sequence of about 40-50 bases within the target nucleic acid. The capture probe sequence is usually complementary to the 3' region of the target nucleic acid (although the reverse, i.e., the 5' region of the target nucleic acid, can also be true), with a melting temperature (T M) is designed to have. The capture probe can be modified at either the 3' or 5' end with a disulfide linker for attachment by covalent bonding to a gold electrode surface, for example, as described in U.S. Patent Nos. 6,753,143 and 7,820,391, each of which is incorporated herein by reference in its entirety, and owned by essentially the same applicant.

[0113] A capture probe, for example, containing nucleic acid, can be adhered directly or indirectly, covalently or non-covalently, to the surface of an electrode or other substrate using a variety of well-known techniques. See, for example, Ch. 13, Chemically Modified Electrodes, Martin and Foss, pp. 403 - 442, Laboratory Techniques in Electroanalytical Chemistry; 2d Ed., Kissinger and Heineman, Eds., MARCEL DEKKER, INC. (1996); Biochip Technology, Cheng and Kricka, Eds. George H. Buchanan Printing Company, Bridgeport, N.J. (2001). In some embodiments, this is done by mixing a disulfide self-assembled monolayer insulator array precursor with the above-described 3' or 5' modified nucleic acid capture probe having a disulfide group and spotting it onto a gold or gold-plated electrode. This is mediated by a linker / functional group, for example, W330 as referenced and described in U.S. Patent No. 7,820,391, which is incorporated by reference in its entirety, or N150 as referenced and described in U.S. Patent No. 6,753,143, which is incorporated by reference in its entirety. As will be understood by those skilled in the art, there are many types of linkers available, for example, as described in the previously referenced literature.

[0114] Signal primer synthesis The signal primer sequence(s) is / are complementary to the specific region(s) of the target. When array discrimination is required, the array polymorphism must be as close as possible to the center of the amplicon array, and the Ts of the two amplicons M must match as precisely as possible.

[0115] When preparing the signal primer, the electron transfer moiety (ETM) can be covalently attached to the nucleic acid at various positions, namely, the 5'-end (Figure 2c), the center of the array (Figure 2b), or both (Figure 2a). In one embodiment, the attachment is via attachment to the base of the nucleoside or to the backbone of the nucleic acid including either the ribose or phosphate moiety of the ribose-phosphate backbone. In embodiments, the composition is designed such that the electron transfer moiety is as close as possible to the "π-way".

[0116] In some embodiments, the signal primer is designed to have a ferrocene label (e.g., N6, QW56, QW80, etc.). Attachment of the ferrocene label should not disrupt the Watson-Crick base pairing of the primer to which the electron transfer moiety is attached and should not interfere with the annealing of the primer to the target. In other embodiments, the primer may contain extra terminal nucleosides at the ends of the nucleic acid (n+1 or n+2), which are used to covalently attach the electron transfer moiety (ETM), but do not participate in base pair hybridization as shown in FIGS. 2j and 2k. In still other embodiments, as shown in FIGS. 2I and 2m, it may be desirable to insert a linker arm that separates the electron transfer moiety from the primer region. In still other embodiments, as shown in FIG. 2n, it may be desirable to have two or more signal moieties on the primer region. In some embodiments, one ferrocene label is added to the signal primer sequence. In some embodiments, two ferrocene labels are added to the signal primer sequence. In some embodiments, three ferrocene labels are added to the signal primer sequence. In some embodiments, four ferrocene labels are added to the signal primer sequence. In some embodiments, five ferrocene labels are added to the signal primer sequence. In some embodiments, six ferrocene labels are added to the signal primer sequence. In some embodiments, seven ferrocene labels are added to the signal primer sequence. In some embodiments, eight ferrocene labels are added to the signal primer sequence. In some embodiments, nine ferrocene labels are added to the signal primer sequence. In some embodiments, ten ferrocene labels are added to the signal primer sequence. In some embodiments, 1 to 10 ferrocene labels are added to the signal primer sequence. In some embodiments, 1 to 6 ferrocene labels are added to the signal primer sequence. In some embodiments, six ferrocene labels are added per signal primer.

[0117] In some embodiments, the signal primer is designed to have a ferrocene label (e.g., N6, QW56, or QW80, etc.) attached to the 5'-end of the non-phosphorylated oligonucleotide signal primer or near the 5'-end (e.g., attached to any one of nucleotides 1 to 3 of the 5'-end of the signal, or attached to any one of nucleotides 1 to 2 of the 5'-end). In some embodiments, the ferrocene label(s) is / are added to the 5'-end of the signal primer sequence(s). In some embodiments, one ferrocene label is added to the 5'-end of the signal primer sequence. In some embodiments, two ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, three ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, four ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, five ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, six ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, seven ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, eight ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, nine ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, ten ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, 1 to 10 ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, 1 to 6 ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, six ferrocene labels are added per signal primer. Since all hybridization reactions must be carried out at a single temperature, the T of all signal primers MThe value must be within a range of 5°C. Since all detection reactions must be carried out in the same solution, the signal primer and the capture probe must be designed to avoid any cross-hybridization, and the maximum ΔG0 value of cross-hybridization has been established empirically.

[0118] The signal primer is synthesized using standard phosphoramidite chemistry and can contain any nucleotide or modified base suitable for DNA polymerase. The nucleic acid portion of the signal primer can be DNA or RNA or a chimeric mixture or derivative or a modified version thereof. In addition to being labeled with an electrochemically detectable label, the signal primer can be modified at the base portion, sugar portion or phosphate backbone and can contain other additional groups or labels.

[0119] The signal primer can be of any suitable size, but ideally does not bind to the capture probe without extension. In some embodiments, the signal primer(s) are in the range of 10 to 100 nucleotides or 10 to 80 nucleotides or 11 to 40 nucleotides or 17 to 25 nucleotides, but the signal primer(s) can be longer or shorter as needed.

[0120] The signal transduction portion of the signal primer can include at least one or more electron transfer moieties selected from the group including but not limited to ferrocene and ferrocene derivatives. In one embodiment, the signal transduction portion of the signal primer can include 1 to 6 electron transfer moieties. In one embodiment, the signal transduction portion of the signal primer can include 1 to 10 electron transfer moieties.

[0121] In some embodiments, the signal primer or single-stranded signal amplicon or double-stranded signal amplicon may include one or more labels (which may be the same or different). In one aspect, the oligonucleotide is labeled, and the label is any moiety that undergoes a detectable electrochemical change upon hybridization with a capture probe on the electrode surface. In one embodiment, the label is a ferrocene tag, and the label undergoes a detectable change in one or more electrochemical properties. Such properties include, but are not limited to, signal intensity, electrochemical potential, or reaction constant.

[0122] The signal transduction portion of the signal primer can be located at one or more positions within the signal primer and / or at or near the 5' end. In one embodiment, the signal transduction portion of the signal primer can be located at the first or second 3' or 5' terminal nucleotide, the first, second, or third 3' or 5' terminal nucleotide, one of the first five 3' or 5' terminal nucleotides, one of the first ten 3' or 5' terminal nucleotides, one of the first fifteen 3' or 5' terminal nucleotides, or one of the first twenty 3' or 5' terminal nucleotides. In certain embodiments, the signal transduction portion of the signal primer is located on the first base at the 3' or 5' end.

[0123] The signal primer lacks an enhancing group. They do not undergo a detectable change in an observable property upon hybridization and / or extension. The signal transduction portion of the signal primer is not a fluorescent moiety.

[0124] The signal primer can be labeled (as described above) using any known labeling method. As an example, the signal primer can be labeled by: (1) attachment at the sulfur of phosphorothioate linkages; (2) attachment at the 2'-amino group; (3) attachment at the 1'-position using appropriately modified sugars, for example, alkylamine-substituted carboxamides; (4) attachment at the 1'-position using, for example, abasic sites and, for example, an alkyldiamine as a linker; (5) generation of a structure by reductive alkylation of an adduct formed between an alkyldiamine and an abasic site; (6) incorporation using 4'-thio-2'-deoxyuridine or 4'-thiothymidine; (7) attachment at the 2'-position of 4-thiothymidine or 4-thio-2'-deoxyuridine; (8) attachment at the 4-amino position of deoxycytidine when the 4-amino group is derivatized with an alkylamine; (9) attachment via the 6'-position of adenine when the 6-amino group is derivatized with an alkylamine moiety; (10) incorporation of adenine using this position when the 8'-position is substituted with an alkylthioamine; (11) attachment at N2 of guanine when the N2 amino is derivatized with an alkylamine group; or (12) attachment at the N2 position of aminoadenine when the 2-amino group is derivatized with an alkylamine.

[0125] All signal primers can be purified using techniques known in the art.

[0126] False positive results False positive results can cause serious problems, including unnecessary use of antibiotics, antibacterial agents, or antifungal treatments. To avoid false positive results, the signal primer should not bind to the capture probe. For example, in FIG. 6, portion c is the extended portion of the amplicon, portion b is the primer region, and portion a is the signal transduction portion. Portion b cannot bind to the capture probe, thus reducing false positives. Further, in the method step, a separation step can be used to separate the signal primer from the single-stranded signal amplicon.

[0127] False negative results False negative results are rarer. In prior art systems, amplicons bind to signal probes. If there are errors in the function or design of the signal probes, false negatives occur. False negatives can be reduced by eliminating the need for amplicons to bind to signal probes.

[0128] Signal primer Unlike capture probes used in the system, signal primers are not bound to the electrodes prior to detection. During detection, instead of binding directly to the electrodes, they bind to capture probes that hold them in place for detection.

[0129] Unlike signal probes used in prior art systems, signal primers are involved in amplification and detection.

[0130] In some embodiments, the primers are made of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid, hexa-polyethylene glycol-modified nucleic acid, chimeric mixtures or derivatives or modified versions thereof.

[0131] In some embodiments, the signal primer does not overlap with the capture probe. In some embodiments, the signal primer overlaps with the capture probe by about 1 to 10 base pairs. In some embodiments, the signal primer overlaps with the capture probe by about 1 to 12 base pairs.

[0132] The signal primers disclosed herein can be better understood by the following embodiments. In one embodiment, the signal primer comprises a nucleic acid and a detectable label. In some embodiments, the signal primer comprises a nucleic acid and an electron transfer moiety. In certain embodiments, the signal primer comprises a nucleic acid and a ferrocene label. In another embodiment, the signal primer comprises a nucleic acid and an electron transfer moiety, and the electron transfer moiety is attached to the 5' end of the primer. In some embodiments, the signal primer comprises a nucleic acid and a label capable of generating a signal during redox-mediated electron detection. In some embodiments, the signal primer comprises a nucleic acid and an electrochemically detectable label. In some embodiments, the signal primer comprises 5 to 12 nucleic acids and an electrochemically detectable label. In some embodiments, the signal primer comprises 5 to 25 nucleic acids and an electrochemically detectable label. In some embodiments, the signal primer is a self-annealing primer and comprises an electrochemically detectable label. In some embodiments, the signal primer comprises a nucleic acid and an electron transfer moiety, the electron transfer moiety is attached to the 5' end of the primer, and is capable of generating a signal during redox-mediated electron detection. In some embodiments, the signal primer comprises a nucleic acid and an electrochemically detectable label, and the electrochemically detectable label is attached to the 5' end of the primer. In some embodiments, the signal primer

[0133] consists of a nucleic acid and an electrochemically detectable label.

[0134] Determination of target concentration When electrochemical detection is used, the main purpose of the nucleic acid amplification step is to generate detectable nucleic acids at a concentration of about 0.01 picomoles from the target molecule, since it is known that this is within the range of the lower limit of detection for electrochemical detection. As is known, if 1 microliter of blood contains about 5×10 3 molecules of DNA, 1 milliliter, a reasonably obtainable sample volume, contains 5×10 6 molecules, or about 107 It contains molecules. To obtain 0.01 pmol of DNA from the amount of DNA in 1 ml of blood, approximately 10 3 times amplification is required. This can surely be achieved using several well-known amplification techniques. It will be apparent to those skilled in the art to perform similar calculations for different sample types and sample volumes to determine the amplification factor.

[0135] Removal of primers after amplification As a result of attempting to develop a system incorporating a rapid PCR reaction, i.e., a system in which amplification is completed in less than about 10 minutes, it was found that it was necessary to increase the primer concentration. As described above, if excess primers are present in the mixture during detection, this can generate a "primer background" in the detection step and may interfere with or reduce the signal generation of single-stranded signal amplicons bound to the capture oligonucleotides.

[0136] Co-owned U.S. Patent No. 10,864,522, which is incorporated herein by reference in its entirety, describes a processing cartridge and method for detecting pathogens in a sample. Specifically, Patent 10,864,522 describes moving a sample, primers, and amplicons on a digital microfluidic pathway. In such a system, the primers and amplicons have different charge-to-mass ratios so that they can be separated from each other by moving them along the digital microfluidic pathway. The PCR double-stranded amplification material moves through the digital microfluidic pathway, the small synthetic oligonucleotide primers move the fastest, and the large amplicons move more slowly. After a certain time, the PCR-amplified material can be split on the digital microfluidic pathway using half of the oligonucleotide primers and the other larger amplicons. In some embodiments, the splitting occurs after the PCR amplification material is denatured with an endonuclease. In one embodiment, a membrane or gel that slows down larger molecules compared to shorter ones can be added to the digital microfluidic pathway to assist in splitting the material.

[0137] Detectable label The detectable label can include an electron transfer moiety. The electron transfer moiety includes a ferrocene label. In some embodiments, the ferrocene label (e.g., N6, QW56, or QW80, etc.) is at the 5' end of the primer or near the 5' end of the primer (e.g., attached to any one of nucleotides 1 to 3 at the 5' end, or attached to any one of nucleotides 1 to 2 at the 5' end). However, the ferrocene label can also be present at both the 3' end and the 5' end of the primer. The signal primer produces a double-stranded signal amplicon. When the double-stranded signal amplicon denatures, it forms a single-stranded signal amplicon that can bind to the capture probe to generate an electrochemical signal. Thus, the signal primer plays a role in both amplification and detection. The signal primer allows the system to utilize shorter signal amplicons. In some embodiments, the amplicon is about 60 base pairs. In some embodiments, the amplicon is about 30 - 120 base pairs. In some embodiments, the amplicon is about 70 - 250 base pairs. In some embodiments, the amplicon is about 50 - 500 base pairs.

[0138] In one embodiment, the nucleic acid is modified with at least one electron transfer moiety at one location. In one embodiment, the nucleic acid is modified with at least two electron transfer moieties at two locations (see Fig. 2n). In one embodiment, the nucleic acid is modified with three or more electron transfer moieties at three or more locations. In one embodiment, the nucleic acid is modified with a plurality of electron transfer moieties at a plurality of positions. For example, a plurality of electron transfer moieties at a plurality of locations may be used to increase the signal obtained from the primer. For example, the electron transfer moiety can be attached to both the 5' and 3' ends as generally shown in Fig. 2n. In one embodiment, the plurality of electron transfer moieties are the same, resulting in a uniform signal. In another embodiment, each of the plurality of electron transfer moieties may be different. In one embodiment, two electron transfer moieties are the same, resulting in a uniform signal. In another embodiment, the two electron transfer moieties may be different.

[0139] In some embodiments, the detectable label is held one base pair away from the capture probe and still generates a detectable signal. In some embodiments, the detectable label is held at any location from 1 to 10 base pairs away from the capture probe and still generates a detectable signal. In some embodiments, the detectable label is held at any location from 1 to 50 base pairs away from the capture probe and still generates a detectable signal. In some embodiments, the detectable label is held at any location from 1 to 100 base pairs away from the capture probe and still generates a detectable signal. In some embodiments, the detectable label is held at any location from 36 to 72 base pairs away from the capture probe and still generates a detectable signal. Thus, provided herein is a capture probe signal primer complex in which an ETM (e.g., ferrocene label, etc.) is 1 to 10 base pairs away from the position where the capture probe hybridizes. In some embodiments, a capture probe signal primer complex is provided in which an ETM (e.g., ferrocene label, etc.) is 1 to 50 base pairs away from the position where the capture probe hybridizes. In a further embodiment, provided herein is a capture probe signal primer complex in which an ETM (e.g., ferrocene label, etc.) is 1 to 100 base pairs away from the position where the capture probe hybridizes. In a further embodiment, provided herein is a capture probe signal primer complex in which an ETM (e.g., ferrocene label, etc.) is 36 to 72 base pairs away from the position where the capture probe hybridizes.

[0140] Single-stranded signal amplicon A single-stranded signal amplicon is shown in FIGS. 2(d - f), where "ii" is the detection part, for example, ferrocene, and "i" is the annealing region, for example, the region that binds to the capture probe during detection. In some embodiments, the amplicon includes two regions, one of which can bind to the capture probe and the other can generate a signal during redox-mediated electron detection. In some embodiments, the amplicon includes three regions, one of which can bind to the capture probe, the second region can generate a signal during redox-mediated electron detection, and the third region connects the first and second regions.

[0141] In the presence of a DNA polymerase with appropriate target and related components, PCR is carried out using the disclosed synthetically labeled oligonucleotide primers, generating newly synthesized DNA molecules incorporating the signal sub-region. This molecule can then be denatured and hybridized to the target sequence on a solid support. The signal sub-region can then be used to generate a signal. In some embodiments, the signal part is a ferrocene label.

[0142] In some embodiments, the single-stranded signal amplicon is 60 bases in length. In some embodiments, the single-stranded signal amplicon is 30 - 60 bases in length. In some embodiments, the single-stranded signal amplicon is 20 - 100 bases in length. In some embodiments, the single-stranded signal amplicon is 20 - 150 bases in length.

[0143] In some embodiments, the amplicon is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid, and hexa-polyethylene glycol-modified nucleic acid.

[0144] In some embodiments, the single-stranded signal amplicon comprises a nucleic acid and a detectable label. In some embodiments, the single-stranded signal amplicon comprises a nucleic acid and an electron transfer moiety. In some embodiments, the single-stranded signal amplicon comprises a nucleic acid and a ferrocene label. In some embodiments, the single-stranded signal amplicon comprises a nucleic acid and an electron transfer moiety, the nucleic acid comprising a first portion and a second portion, the first and second portions being different.

[0145] In some embodiments, the single-stranded signal amplicon includes a nucleic acid and an electron transfer moiety, the nucleic acid includes a first portion and a second portion, the first portion and the second portion are different, the first portion can bind to a first capture probe, and the second portion can bind to a second capture probe. In some embodiments, the single-stranded signal amplicon includes a nucleic acid and an electron transfer moiety, the nucleic acid includes a first portion and a second portion, the first portion and the second portion are different, the first portion can bind to a first capture probe but cannot bind to a second capture probe, and the second portion can bind to a second capture probe but cannot bind to a first capture probe. In some embodiments, the single-stranded signal amplicon includes a nucleic acid and an electron transfer moiety, the nucleic acid includes a first portion and a second portion, the first portion and the second portion are different, the first portion can bind to a first capture probe and a second capture probe. In some embodiments, the single-stranded signal amplicon includes a nucleic acid and an electron transfer moiety, the nucleic acid includes a first portion and a second portion, the first portion and the second portion are different, the first portion can bind to a first capture probe and a second capture probe, and the second portion can bind to a first capture probe and a second capture probe. In certain embodiments, the electron transfer moiety is attached to the 5'-end or near the 5'-end of the single-stranded signal amplicon (e.g., attached to any one of nucleotides 1 to 3 at the 5'-end, or attached to any one of nucleotides 1 to 2 at the 5'-end). In some embodiments, the single-stranded signal amplicon includes an electrochemically detectable label. In some embodiments, the single-stranded signal amplicon includes a capture probe binding region, a primer binding region, and an electrochemically detectable label region. Here, in certain embodiments, the electrochemically detectable label region is 5 to 100 base pairs away from the capture probe binding region. In some embodiments, the electron transfer moiety is an electrochemically detectable label. Here, in certain embodiments, the electron transfer moiety is an electrochemically detectable label and is attached to the 5'-end or near the 5'-end of the single-stranded signal amplicon.In some embodiments, the single-stranded signal amplicon consists of a capture probe binding region, a primer binding region, and an electrochemically detectable label region. Here, in certain embodiments, the electrochemically detectable label is 5 to 100 base pairs away from the capture probe binding region. In some embodiments, the single-stranded signal amplicon includes electrochemically detectable labels attached to the 5' and 3' ends of the single-stranded signal amplicon.

[0146] Double-stranded amplicon In some embodiments, the double-stranded signal amplicon is 60 base pairs in length. In some embodiments, the double-stranded signal amplicon is 30 to 60 base pairs in length. In some embodiments, the double-stranded signal amplicon is 20 to 100 base pairs in length. In some embodiments, the double-stranded signal amplicon is 20 to 150 base pairs in length.

[0147] In some embodiments, the double-stranded signal amplicon includes a nucleic acid and a detectable label. In some embodiments, the double-stranded signal amplicon includes a nucleic acid and an electron transfer moiety. In some embodiments, the double-stranded signal amplicon includes a nucleic acid and a ferrocene label. In some embodiments, the double-stranded signal amplicon includes a nucleic acid and an electron transfer moiety, the nucleic acid includes a first portion and a second portion, and the first portion and the second portion are different. In some embodiments, the double-stranded signal amplicon includes a nucleic acid and an electron transfer moiety, and the electron transfer moiety is attached to the 5' end or near the 5' end of the double-stranded signal amplicon. In some embodiments, the double-stranded signal amplicon includes a nucleic acid and an electron transfer moiety, and the electron transfer moiety is attached to the forward and reverse ends of the double-stranded signal amplicon. In some embodiments, the double-stranded signal amplicon includes an electrochemically detectable label.

[0148] Hybridization complex: capture probe and signal amplicon In some embodiments, the forward primer and the reverse primer are labeled with an electrochemically detectable label. In some embodiments, the forward primer is labeled with an electrochemically detectable label. In some embodiments, the reverse primer is labeled with an electrochemically detectable label. In some embodiments, there are a first capture probe specific to the forward primer labeled with an electrochemically detectable label and a second capture probe specific to the reverse primer labeled with an electrochemically detectable label.

[0149] In some embodiments, the hybridization complex comprises a capture probe bound to an amplicon, the amplicon comprising a nucleic acid and a detectable label. In some embodiments, the hybridization complex comprises a capture probe bound to an amplicon, the amplicon comprising a nucleic acid and an electron transfer moiety. In some embodiments, the hybridization complex comprises a capture probe bound to an amplicon, the amplicon comprising a nucleic acid and a ferrocene label. In some embodiments, the hybridization complex comprises a capture probe bound to an amplicon, the amplicon comprising a nucleic acid and an electron transfer moiety, the nucleic acid comprising a first portion and a second portion, the first portion and the second portion being different. In some embodiments, the hybridization complex comprises a capture probe bound to an amplicon, the amplicon comprising a nucleic acid and an electron transfer moiety, the nucleic acid comprising a first portion and a second portion, the capture probe being capable of binding to the first portion but not to the second portion. In some embodiments, the hybridization complex comprises a capture probe bound to an amplicon, the amplicon comprising a nucleic acid and an electron transfer moiety, the nucleic acid comprising a first portion and a second portion, the capture probe being capable of binding to the second portion but not to the first portion. In some embodiments, the hybridization complex comprises a capture probe bound to an amplicon, the amplicon comprising a nucleic acid and an electron transfer moiety, the nucleic acid comprising a first portion and a second portion, the capture probe being capable of binding to the second portion and the first portion. In some embodiments, the hybridization complex comprises a capture probe bound to an amplicon, the amplicon comprising a nucleic acid and an electron transfer moiety, the hybridization complex being bound to an electrode, the electrode comprising a monolayer.

[0150] Method The signal primers disclosed herein can be used in diagnostic methods, and the signal primers are complementary to the sequences of infectious disease agents (e.g., genomes or cDNAs), or can initiate the synthesis or amplification of sequences of human diseases including, but not limited to, infectious disease agents such as viruses (e.g., HIV, HPV, etc.), bacteria, parasites, and fungi, thereby diagnosing the presence of infectious agents in a sample from a patient. The type of target nucleic acid can be genomic, cDNA, mRNA, or synthetic, or the source can be human, animal, fungal, or bacterial. In another embodiment that can be used for the diagnosis or prognosis of a disease or disorder, the target sequence can be wild-type human genomic DNA or RNA, or cDNA sequence, or a mutant sequence, the mutation of which is involved in the presence of a human disease or disorder. In such an embodiment, the amplification reaction can be repeated for the same sample using different sets of signal primers that selectively identify the wild-type sequence or the mutant (e.g., using signal primers labeled differently). By way of example, the mutation can be an insertion, substitution, and / or deletion, or translocation of one or more nucleotides. In another embodiment, the signal primers can be used in SNP analysis, pharmacogenomics, and toxicogenetics.

[0151] In certain embodiments, a method for detecting or measuring the products of a nucleic acid amplification or synthesis reaction, comprising: (a) contacting a sample containing or suspected of containing one or more target nucleic acid molecules with one or more signal primers (such primers may contain one or more labels, which may be the same or different, and may be labeled internally and / or near the 3'-end or 5'-end), wherein when the target sequence or nucleic acid molecule is present in the sample, the primers are adapted for use in the amplification or synthesis reaction such that the primers are incorporated into the amplification or synthesis products of the amplification or synthesis reaction; (b) performing an amplification or synthesis reaction; and (c) detecting or measuring one or more synthesis or amplification product molecules by redox-mediated electron detection.

[0152] In another embodiment, a method for detecting a target nucleic acid sequence, comprising contacting a sample containing a mixture of nucleic acids with at least one oligonucleotide capable of hybridizing to the target nucleic acid sequence and containing at least one detectable moiety, wherein the detectable moiety undergoes a redox reaction after an electrical signal is applied, and a change in the redox potential indicates the presence of the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is not separated from the mixture.

[0153] In another embodiment, a method for quantifying a target nucleic acid molecule, comprising contacting a sample containing a mixture of nucleic acids containing the target nucleic acid molecule with at least one oligonucleotide containing a detectable moiety, wherein the detectable moiety undergoes a detectable redox reaction upon application of a charge, and observing a reaction, wherein the observable reaction is proportional to the amount of the target nucleic acid molecule in the sample.

[0154] In another embodiment, a method for amplifying one or more nucleic acid molecules, comprising: (a) mixing one or more template or target nucleic acid molecules with one or more signal primers; and (b) incubating the mixture under conditions sufficient to synthesize or amplify one or more nucleic acid molecules complementary to all or a portion of the template or target molecule, is disclosed. In one embodiment, the amplified nucleic acid molecule comprises one or more signal primers. In one aspect, the signal primer is incorporated at or near one or both ends of the produced synthetic or amplified nucleic acid molecule. Also disclosed are one or more nucleic acid molecules produced by such amplification or synthesis reactions.

[0155] In another embodiment, a method for determining the presence or absence of a target in a sample is disclosed. In some embodiments, the target sequence is a wild-type human genomic or RNA or cDNA sequence. In some embodiments, the target sequence is a mutant human genomic or RNA or cDNA sequence. The mutation is involved in the presence of a human disease or disorder. In some embodiments, the signal primer amplifies the wild-type target, and in other embodiments, the mutated target. By way of example, the mutation can be an insertion, substitution and / or deletion of one or more nucleotides, or a translocation. In another embodiment, the signal primer can be used in SNP analysis, pharmacogenomics and toxicogenetics.

[0156] In certain embodiments, a method for detecting the presence or absence of a nucleic acid amplification product, comprising: (a) contacting a sample suspected of containing or containing one or more target nucleic acid molecules with one or more signal primers (such primers may contain one or more labels, which may be the same or different, and may be labeled internally and / or at or near the 3' end and / or at or near the 5' end), wherein the signal primer is adapted for use in the amplification or synthesis reaction such that when the target sequence or nucleic acid molecule is present in the sample, the signal primer is incorporated into the amplification product of the amplification reaction; (b) performing an amplification reaction; and (c) detecting or measuring one or more amplification product molecules by electrochemical detection.

[0157] In another embodiment, a method for determining the absence of at least one specific target or template nucleic acid molecule in a sample, comprising: (a) contacting the sample with a signal primer, wherein at least a portion of the signal primer is capable of forming base pairs (e.g., hybridizing) with at least a portion of the target nucleic acid molecule; and (b) incubating the signal primer and nucleic acid molecule mixture under conditions sufficient to amplify at least a portion of the target nucleic acid molecule. Under such conditions, the absence of amplification indicates the absence of one or more specific nucleotides in the sample.

[0158] In one embodiment, it comprises: (a) combining reagents for polymerase chain reaction, a polymerase (e.g., DNA polymerase), a target nucleic acid, and a modified primer, wherein the modified primer contains a detectable label, combining reagents for polymerase chain reaction, a polymerase (e.g., DNA polymerase), a target nucleic acid, and a modified primer; (b) cycling the mixture of (a) to provide multiple copies of an amplicon incorporating the modified primer; (c) exposing the mixture of (b) to an exonuclease to produce single-stranded amplicons; (d) exposing the mixture of (c) to a capture oligonucleotide complementary to the single-stranded signal amplicon incorporating the modified primer; (e) hybridizing the single-stranded signal amplicon incorporating the modified primer with the capture oligonucleotide; and (f) detecting the label associated with the hybridization. A method for performing a nucleic acid amplification assay is provided. In some embodiments, the target nucleic acid is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, and their modifications and derivatives. In some embodiments, the target nucleic acid is extracted from blood, oral swabs, tissue, body fluids, environmental samples, the surface of materials, plants, animals, bacteria, or fungi. In some embodiments, the polymerase is a DNA polymerase, such as Taq polymerase or Thermococcus kodakiensis polymerase. In some embodiments, the primer is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid, and hexa-polyethylene glycol-modified nucleic acid. In some embodiments, the detectable label is an electron transfer moiety. In some embodiments, the detectable label is a ferrocene label. In certain embodiments, the detectable label is N6, QW56, or QW80. In some embodiments, the cycling is isothermal. In some embodiments, the cycling is between a first temperature and a second temperature. In some embodiments, the capture oligonucleotide is immobilized on a gold surface.In some embodiments, the capture oligonucleotide is immobilized on the electrode surface. In some embodiments, the detection is electrochemical. In some embodiments, the label is not exposed to a second label or a second moiety having a quencher.

[0159] In another embodiment, a method of performing a nucleic acid amplification assay, comprising: (a) combining a PCR reagent, a polymerase (e.g., a DNA polymerase), a target nucleic acid, or a sample suspected of containing the target nucleic acid, a modified primer and a second unmodified primer, wherein the modified primer contains a ferrocene label; (b) cycling the mixture of (a) if the target nucleic acid is present in the sample to provide multiple copies of a first amplicon incorporating the modified primer and a second amplicon incorporating the second unmodified primer; (c) exposing the mixture of (b) to an exonuclease to produce a first single-stranded signal amplicon incorporating the modified primer and a second single-stranded amplicon incorporating the unmodified primer; (d) exposing the mixture of (c) to a capture oligonucleotide complementary to the single-stranded signal amplicon incorporating the modified primer; (e) hybridizing the single-stranded signal amplicon of the amplicon incorporating the modified primer to the capture oligonucleotide; and (f) detecting the label associated with the modified primer incorporated into the amplicon. In some embodiments, the target nucleic acid is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, and modifications and derivatives thereof. In some embodiments, the target nucleic acid is extracted from blood, oral swabs, tissue, body fluids, environmental samples, surfaces of materials, plants, animals, bacteria, or fungi. In some embodiments, the reagent comprises reagents for polymerase chain reaction amplification. In some embodiments, the polymerase is a DNA polymerase and is Taq polymerase. In some embodiments, the polymerase is a DNA polymerase and is Thermococcus kodakiensis polymerase. In some embodiments, the primer is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid, and hexa-polyethylene glycol-modified nucleic acid.In some embodiments, the detectable label is selected from the group consisting of N6, QW56, and QW80. In some embodiments, the detectable label is ferrocene, methylene blue, or osmium. In some embodiments, the cycle is isothermal. In some embodiments, the cycle is between a first temperature and a second temperature. In some embodiments, the capture oligonucleotide is immobilized on a gold surface. In some embodiments, the capture oligonucleotide is immobilized on an electrode. In some embodiments, a single-stranded signal amplicon incorporating a modified primer hybridizes to the capture oligonucleotide. In some embodiments, the detection is electrochemical.

[0160] Integrated nucleic acid test cartridge Integrated multiplex target analysis systems are known and are described in U.S. Patent No. 10,864,522, which is incorporated herein by reference.

[0161] An integrated nucleic acid test cartridge capable of performing amplification and detection is provided herein. Generally, the integrated nucleic acid test cartridge can receive a sample, extract DNA, combine the DNA with amplification reagents including ETM-labeled primers, amplify the DNA, incubate the signal amplicon with exonuclease to form a single-stranded signal amplicon, and combine the single-stranded signal amplicon with a capture probe and electrical sensor detection.

[0162] In certain embodiments, the target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid and modifications and derivatives thereof, and in other embodiments, the target nucleic acid is extracted from blood, oral swabs, tissue, body fluids, environmental samples, surfaces of materials, plants, animals, bacteria, and fungi.

[0163] An integrated nucleic acid test cartridge capable of performing amplification and detection is further disclosed herein. An integrated nucleic acid test cartridge for use with electrochemical detection is further disclosed herein. An integrated nucleic acid test cartridge capable of performing amplification is further disclosed herein. An integrated nucleic acid test cartridge capable of performing detection is further disclosed herein. An integrated nucleic acid test cartridge capable of performing amplification and detection is further disclosed herein. Herein, an integrated cartridge for nucleic acid testing that operates in conjunction with a reader device is further disclosed.

[0164] Kit A kit for detecting nucleic acid molecules in a sample is disclosed. Such a kit may also be designed to detect the nucleic acid molecule of interest during or after a nucleic acid amplification reaction. Such a kit can be a diagnostic kit in which the presence of the nucleic acid correlates with the presence or absence of a disease or disorder. A kit for performing the amplification reaction described herein and a kit for preparing the compositions described herein are disclosed.

[0165] In certain embodiments, the kit includes one or more signal primers as defined herein. The kit can further include additional components for performing a detection assay or other method.

[0166] Such a kit can include one or more additional components selected from the group consisting of one or more polymerases (e.g., DNA polymerase and reverse transcriptase), one or more nucleotides, one or more buffer salts (including nucleic acid amplification buffer), one or more control nucleic acid target molecules (to act as a positive control for testing the assay), instructions for performing the method, and the like.

Example

[0167] Example 1: Amplification and Detection Using Signal Primers SARS-CoV-2 amplicons were amplified by either control primers or signal primers (Figure 10). In this example, a reverse primer for the SARS-CoV 2 N1 gene with an N6 tag was synthesized. A tagless simultaneous reverse primer was used as a control. After digestion with λ exonuclease, the "control" amplicons were incubated with the SARS-CoV-2 N1 signal probe. On the other hand, the amplicons generated using the signal primers were digested with λ exonuclease but not incubated with the signal probe. The amplicons were then loaded into the detection zone of the GenMark Respiratory Panel cartridge (Carlsbad, CA). The signal generated from the amplicons using the signal primers was higher than the signal generated using the control primers (Figure 10). Furthermore, no false-positive signals were detected from other targets within the detection zone, confirming that the signal primers did not bind nonspecifically to the capture probe (Figure 11).

[0168] Example 2: Detection using signal primers vs. conventional sandwich assay using ferrocene away from the capture probe (control primers) Adenovirus C, OC43, RSV A, and SARS-CoV-2 were amplified by either a current reverse primer without a tag (“control primer”) or the corresponding signal primer. The base distance between the ferrocene label and the capture probe is shown in Figure 12. In Figure 6, this is the distance shown in part b. After digestion with exonuclease λ, the “control” amplicons were incubated with their respective signal probes. Amplicons generated by λ using the signal primer were not digested with exonuclease and were not incubated with the signal probe. The amplicons were then loaded into the detection zone of the GenMark Respiratory Panel cartridge (Carlsbad, CA). The signal generated from the amplicons using the signal primer was generally greater than the signal generated using the control primer (Figure 12) for various targets.

Claims

1. A method for detecting the presence or absence of a target nucleic acid in a sample, wherein the method is a. A solution containing or suspected to contain a target nucleic acid is combined with an amplification reagent to amplify the target nucleic acid if present, wherein the amplification reagent includes an electrochemically detectable label and a signal primer containing a nucleic acid sequence complementary to at least a portion of the target nucleic acid, and thereby capable of hybridizing to the target nucleic acid. b. If the target nucleic acid is present, amplify the target nucleic acid to produce a double-stranded labeled amplicon, c. Incubating the double-stranded labeled amplicon with an exonuclease to form a single-stranded labeled amplicon, d. Hybridizing the single-strand labeled amplicon with the first capture probe, e. Detecting the presence or absence of the target nucleic acid in the sample using electrochemical detection. Methods that include...

2. The method according to claim 1, wherein the signal primer, the double-stranded labeled amplicon, and / or the single-stranded labeled amplicon comprises at least one label selected from the group consisting of ferrocene, methylene blue, or osmium.

3. The signal primer contains a ferrocene label at its 5' end, one of the nucleotides 1-3 at the 5' end, at its 3' end, one of the nucleotides 1-3 at the 3' end, or at both its 5' and 3' ends, or The method according to claim 1 or 2, wherein the signal primer includes a ferrocene label inside the signal primer.

4. The method according to claim 1 or 2, wherein the signal primer, the double-stranded labeled amplicon, and / or the single-stranded labeled amplicon include a plurality of ferrocene labels at at least two locations.

5. The method according to claim 1 or 2, wherein the single-stranded labeled amplicon comprises a first section that can hybridize to the first capture probe, a second section that cannot bind to the capture probe, and a third section that includes at least one label.

6. The method according to claim 1 or 2, wherein the signal primer comprises a first section that can hybridize to the first capture probe, a second section including a linker, and a third section including at least one label, the linker connecting the first section and the third section.

7. The method further includes separating the signal primer and the single-stranded labeled amplicon before electrochemical detection, or The method according to claim 1 or 2, further comprising separating the labeled primer and the double-stranded labeled amplicon before electrochemical detection.

8. The method according to claim 1 or 2, wherein the signal primer comprises a first section that can hybridize to the capture probe and a second section that includes at least a first label.

9. A method for detecting the presence or absence of a target nucleic acid in a sample, wherein the method is a. Receiving the sample, b. Extracting nucleic acids from the sample, wherein the nucleic acids are suspected to contain target nucleic acids, c. Amplifying the nucleic acid in combination with an amplification reagent, if the target nucleic acid is present, wherein the amplification reagent includes an electrochemically detectable label and a signal primer containing a nucleic acid sequence complementary to at least a portion of the target nucleic acid, and thereby a signal primer capable of hybridizing to the target nucleic acid; d. Amplifying the target nucleic acid to produce a double-stranded signal amplicon, if the target nucleic acid is present; e. Incubating the double-stranded signal amplicon with an exonuclease to form a single-stranded signal amplicon, f. Hybridizing the single-stranded signal amplicon with the first capture probe, g. Detecting the presence or absence of the target nucleic acid in the sample using electrochemical detection. Methods that include...

10. The method according to claim 9, wherein the signal primer comprises at least one label selected from the group consisting of ferrocene, methylene blue, or osmium.

11. The method according to claim 9 or 10, wherein a first portion of the single-stranded signal amplicon is coupled to the first capture probe, and a second portion of the single-stranded signal amplicon is coupled to the second capture probe.

12. A method for detecting the presence of a target single-stranded or double-stranded nucleic acid in a sample, wherein the method is a. i. Samples suspected of containing the target nucleic acid mentioned above, ii.

1. A nucleic acid sequence complementary to at least a portion of the target nucleic acid, 2. A signal primer comprising a first electrochemically detectable label, iii. Reagents for nucleic acid chain elongation The process of providing, b. A step of forming a reaction mixture including (i), (ii) and (iii) above, c. A step of contacting the signal primer with the target nucleic acid, if present, under hybridization conditions, d. A step of extending the signal primer and thereby incorporating the electrochemically detectable label into the amplicon, and if the target nucleic acid is present, forming an electrochemically labeled amplicon; e. A step of modifying the electrochemically labeled amplicon, f. A step of hybridizing the electrochemically labeled amplicon with a capture probe bonded to the electrode surface, g. A step of detecting the presence of the target nucleic acid by detecting the energy transfer between the electrochemically labeled amplicon and the electrode surface. Methods that include...

13. The method according to claim 12, wherein the electrochemically detectable label is selected from the group consisting of ferrocene, methylene blue, or osmium.

14. The method according to claim 12 or 13, wherein the signal primer includes a first portion that binds to the capture probe and a second portion that does not bind to the capture probe.

15. The method according to claim 12 or 13, wherein the signal primer further comprises a second electrochemically detectable label, the first electrochemically detectable label and the second electrochemically detectable label being different.

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