WO2022096893A1 - Dispositif - Google Patents

Dispositif Download PDF

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Publication number
WO2022096893A1
WO2022096893A1 PCT/GB2021/052870 GB2021052870W WO2022096893A1 WO 2022096893 A1 WO2022096893 A1 WO 2022096893A1 GB 2021052870 W GB2021052870 W GB 2021052870W WO 2022096893 A1 WO2022096893 A1 WO 2022096893A1
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WO
WIPO (PCT)
Prior art keywords
probe
polynucleotide
analyte
subject
fluid sample
Prior art date
Application number
PCT/GB2021/052870
Other languages
English (en)
Inventor
Melissa Grant
Tim Albrecht
Oliver IRVING
Original Assignee
The University Of Birmingham
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB2017465.2A external-priority patent/GB202017465D0/en
Priority claimed from GBGB2104219.7A external-priority patent/GB202104219D0/en
Application filed by The University Of Birmingham filed Critical The University Of Birmingham
Priority to US18/035,124 priority Critical patent/US20230408508A1/en
Priority to EP21814839.3A priority patent/EP4241084A1/fr
Priority to EP22712620.8A priority patent/EP4314820A1/fr
Priority to PCT/GB2022/050735 priority patent/WO2022200793A1/fr
Publication of WO2022096893A1 publication Critical patent/WO2022096893A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56955—Bacteria involved in periodontal diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present invention relates to a system and a device for use in the diagnosis of an oral disease.
  • Methods, kits and compositions for use in the diagnosis of oral disease are also provided. More particularly, the invention relates to a system comprising a probe which is configured to collect a fluid sample from the oral cavity of a subject, and a detector which is configured to detect in the fluid sample the presence and/or concentration of an analyte which is indicative of the oral disease.
  • Periodontal disease also known as gum disease
  • gingivitis The early stage of the disease is known as gingivitis, the symptoms of which include inflammation of the gums, which may bleed after brushing. This inflammation is usually a response to bacterial biofilms or plaques which have formed on teeth. If left untreated, gingivitis can progress to a more serious condition known as periodontitis, the symptoms of which may include loose teeth and gum abscesses.
  • Periodontal disease has also been associated with a number of other conditions including lung infections, cardiovascular disease and premature labour.
  • Periodontal disease is the leading cause of tooth loss in adults, and is believed to affect -10% of the population worldwide, and is particularly prevalent in people over the age of 65.
  • a dentist will typically look for a number of indicators including tooth movement, sensitivity, gum bleeding, swelling and pocket depth. Large pocket depths around teeth are an indicator of gum disease. However, pocket depth is only a secondary indicator in that it indirectly indicates the presence of disease. Furthermore, the depth of the pocket does not provide any information about the progression of the disease, and thus what type of therapy is most appropriate.
  • WO2014/037924 describes analysis of biomarkers of gingivitis and periodontitis using Fourier Transform - tandem Mass Spectrometry (FT MS/MS).
  • FT MS/MS Fourier Transform - tandem Mass Spectrometry
  • WO2019141525 and WO2019141547 describe the detection of biomarkers of periodontal disease using mass spectrometry, infrared or immunological assays. Mass spectrometry and infrared analysis again require expensive equipment and specialist training. Immunological assays are time consuming and can only be carried out by a person skilled in molecular biology techniques. Furthermore, assays such as ELISAs require numerous reagents including multiple antibodies.
  • a system for diagnosing an oral disease in a subject comprising: a probe which is configured to collect a fluid sample from the oral cavity of the subject; and a detector which is configured to detect in the fluid sample the presence and/or concentration of an analyte which is indicative of the oral disease.
  • the detector may be configured to receive the fluid sample from the probe.
  • the probe and the detector are separate components.
  • the detector is in fluid communication with the probe i.e. the detector is coupled to the probe.
  • the probe may be coupled to the detector via a conduit, e.g. a tube or capillary, which enables transfer of the fluid sample from the probe to the detector.
  • the detector may be coupled to the probe permanently or semi-permanently, or temporarily e.g. only during transfer of the fluid sample from the probe to the detector.
  • the probe is not coupled to the detector.
  • the fluid sample may be deposited (e.g. manually) from the probe into a portal, opening or receptacle, from which the fluid sample is transferred to the detector.
  • the detector and the probe are integrated into a single device.
  • both the probe and the detector may be contained within the same housing.
  • a device for use in diagnosing an oral disease in a subject comprising a probe which is configured to collect a fluid sample from the oral cavity of the subject.
  • the device further comprises a detector which is configured to detect in the fluid sample the presence and/or concentration of an analyte which is indicative of the oral disease.
  • the probe or the device may be hand-held.
  • the system and device of the invention enable both local fluid sampling from specific sites within the mouth and, optionally, integrated analysis. This provides the ability to detect analytes indicative of periodontal disease with local spatial resolution. For example, analyte concentrations may be mapped for each individual tooth, thereby providing site-specific determination of disease progression, and in turn enabling therapy to be targeted appropriately.
  • analytes e.g. biomarkers
  • the integration of the probe and detector into a single system or device conveniently enables in situ detection and/or quantification of analytes (e.g. biomarkers), which can be carried out by a dentist or dental hygienist, rather than having to send a sample to a laboratory for analysis.
  • the detector is configured to detect the presence and/or concentration of the analyte using resistive pulse sensing (RPS).
  • RPS resistive pulse sensing
  • RPS can be adopted for identifying analytes (e.g. biomarkers) indicative of oral disease in fluid samples taken from the oral cavity (e.g. in saliva and/or GCF).
  • RPS is suitable for both detecting the presence of analytes and their abundance, and thus can be used to measure local analyte concentration. Since RPS provides an electrical signal, the results of RPS detection are rapidly provided.
  • the use of RPS thus enables real-time detection of analytes, significantly reducing the time required to analyse a fluid sample compared to traditional assays, such as ELISA. Since the results are available quickly, patients can be diagnosed and treated within a single appointment. This enhances the patient experience and reduces cost through a more efficient use of time and resources.
  • the cost of RPS analysis is low since few reagents are required, and can be carried out with less specialist knowledge and training compared to techniques such as mass spectrometry.
  • the detector comprises a nanopore.
  • resistive pulse sensors can be used as single-molecule detectors.
  • resistive pulse sensors comprise a single, well-defined nanoscale pore embedded in a membrane which separates two electrolyte-filled compartments, each containing one electrode.
  • the application of a voltage between the electrodes results in an ion current, which in turn leads to potential drops and local electric fields in the cell.
  • Suitably strong electric fields can pull charged objects in solution toward and eventually through the nanopore. Because the nanopore normally constitutes the largest source of resistance in the cell, such a translocation event can cause a measurable ion current modulation, thereby enabling detection of molecules in solution.
  • the nanopore is a solid state nanopore.
  • Aa solid state (i.e. chip-based) nanopore typically comprises a pore in a membrane.
  • the membrane may be formed from a material such as Si2N4, SiC>2, graphene or M0S2.
  • the nanopore is a biological nanopore, e.g. a nanopore formed within a protein.
  • Biological nanopores may be embedded in a lipid bilayer.
  • the nanopore is sized so as to allow the passage of a single analyte, or a single carrier molecule with one or more analytes bound thereto, through the pore at a time.
  • the size of the nanopore may be selected by the skilled person in accordance with the size of the analyte and/or the size of the carrier molecule.
  • the pore size may be comparable to, or slightly larger than, the maximum dimension of the analyte.
  • the nanopore may be at least 5, at least 10, at least 20, at least 30, at least 50, at least 70, at least 100 or at least 130 nm in diameter. In some embodiments the nanopore is no more than 200 nm, no more than 150 nm, no more than 100 nm in diameter, no more than 50 nm, no more than 40 nm, or no more than 30 nm in diameter. Suitably, the nanopore may be from 5 to 100, from 10 to 50, from 15 to 40 or from 20 to 30 nm in diameter. It will be appreciated that nanopores are not necessarily circular. As such, the “diameter” of the nanopore in this context refers to the average dimension of the pore. It will further be appreciated that references to the diameter of the nanopore refer to the internal diameter.
  • the nanopore may be provided within a nanopipette.
  • the detector comprises a nanopipette.
  • nanopipettes are a class of nanopores which can be used for the detection and analysis of single molecules in solution. Nanopipettes can be easily fabricated with highly controlled pore sizes, making them a cost effective alternative to traditional solid state nanopores.
  • the detection and analysis of a single molecule using a nanopipette relies on resistive pulse sensing. For this, the nanopipette is filled with and the tip immersed in an electrolyte and a voltage is applied between an electrode in side and an electrode outside of the nanopipette to generate an electric field at the tip. This field drives the molecule of interest through the nanopipette pore, resulting in a detectable pulse.
  • the nanopipette may be formed from any suitable material, such as a metal, polymer, glass, quartz, organic materials (e.g. graphene) or inorganic materials (e.g. boron nitride). In some embodiments, the nanopipette is made from glass or quartz.
  • Nanopipettes may be fabricated using methods which will be known to those skilled in the art, such as those described herein. Typically, nanopipettes are pulled from capillaries (e.g. quartz) using mechanical pipette pullers.
  • capillaries e.g. quartz
  • the device comprises a double-barrel nanopipette, also known as a theta pipette.
  • a double-barrel nanopipette comprises two channels terminating in adjacent nanopores at the tip of the nanopipette, which are separated by a gap of approximately 20 nm (Cadinu et al., Nano Letters 2017, 17, 6376; Cadinu et al., Nano Letters 2018, 18, 2738). T ranslocation of an analyte takes place from one channel to the other.
  • a double barrel nanopipette may function to collect the fluid sample, and as the detector for detecting the presence and/or concentration of an analyte in the fluid.
  • the double-barrel nanopipette may comprise a first barrel (or channel) and a second barrel (or channel).
  • the first channel may be configured to collect a fluid sample from the subject (e.g. from the gingival crevice), for example by means of electroosmosis. Translocation of an analyte present in the fluid sample from the first channel to the second channel generates a detectable signal.
  • the first channel may comprise a first electrode.
  • the second channel may comprise a second electrode.
  • application of a voltage to the first electrode relative to the second electrode may be used to drive a fluid sample into the first channel by electroosmotic flow.
  • applying a positive bias to the second electrode relative to the first electrode may be used to translocate analytes present in the fluid sample from the first channel to the second channel.
  • Translocation of the analytes from the first channel to the second may be carried out after removal of the nanopipette from the mouth of the subject.
  • the fluid sample may be incubated in the first channel with a carrier molecule functionalised with a capture moiety which is capable of specifically binding to a target analyte (e.g. a biomarker) indicative of an oral disease which may be present in the fluid sample.
  • a target analyte e.g. a biomarker
  • Translocation of the target analyte (e.g. biomarker) bound to the carrier molecule from the first channel to the second channel would generate a detectable signal which is distinguishable from the signal generated by carrier molecules not bound to target biomolecules.
  • the probe must be suitably sized and shaped for collecting a fluid sample from the oral cavity of the subject. It will therefore be appreciated that “configured to collect a fluid sample from the oral cavity of the subject” means that the probe is sized and shaped so as to enable sampling from small and/or difficult-to-reach locations. The size and shape of the probe may therefore depend on a number of factors, including the species and/or age of the subject. Suitably, the probe may comprise a curved or bent head. This facilitates the collection of fluid samples from inside the mouth.
  • the probe may be provided with a tip which is configured for collecting a fluid sample. Fluid collection may be achieved by any suitable means, including suction, electroosmosis or capillary action.
  • the tip comprises a capillary.
  • the tip of the probe may be configured for collecting a fluid sample from a periodontal pocket.
  • a periodontal pocket is a space between a tooth and the surrounding gum tissue, caused by the gum pulling away from the tooth. Formation of periodontal pockets is a sign of gum disease. The depth of the pocket is an indicator of the progression of the disease.
  • the tip is tapered.
  • a narrow, tapered shape facilitates insertion of the tip into a periodontal pocket so that a sample of GCF or saliva may be obtained.
  • the tip may have an outer diameter of from 0.3 mm to 2 mm, from 0.4 mm to 1.8 mm, from 0.4 mm to 1.5 mm, from 0.5 mm to 1.4 mm, from 0.6 mm to 1.3 mm, from 0.7 mm to 1.2 mm, from 0.8 mm to 1.1 mm or from 0.9 to 1.0 mm, e.g. approximately 1 mm.
  • the tip may have an outer diameter of from 10 to 300 nm, from 20 nm to 200 nm, from 30 to 180 nm, from 50 nm to 150 nm, from 60 nm to 120 nm or from 80 nm to 110 nm, for example approximately 100 nm.
  • the tip may have an internal diameter of from 2 nm to 200 nm, from 5 nm to 180 nm, from 10 nm to 150 nm, from 20 nm to 120 nm, or from 50 nm to 100 nm. In some embodiments, at its narrowest point the tip has an internal diameter of from about 10 to about 100 nm.
  • the tip and/or the head of the probe is disposable. This helps to avoid contamination between subjects.
  • the probe, or a portion thereof may be formed from any material that is suitable for collecting a fluid sample from the oral cavity of a subject. Suitable materials include metal, plastic, quartz, glass, organic materials (e.g. graphene) or inorganic materials (e.g. boron nitride), or a combination thereof.
  • Suitable materials include metal, plastic, quartz, glass, organic materials (e.g. graphene) or inorganic materials (e.g. boron nitride), or a combination thereof.
  • the probe or the device comprises, or is constituted by, a periodontal probe.
  • a periodontal probe is an instrument commonly used in the field of dentistry, which is primarily used to measure pocket depths around a tooth.
  • a periodontal probe typically has a curved or bent head formed from a thin strand of material (e.g. metal), usually of circular crosssection, having a narrow, tapered tip with distance markings thereon.
  • a periodontal probe has a solid tip.
  • the periodontal probe is configured for collecting a fluid sample from the oral cavity of the subject. Accordingly, the tip of the periodontal probe is hollow, or has a capillary therethrough.
  • the tip of the probe has one or more markings on an outer surface thereof.
  • the markings may be provided at a distance of 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm and/or 10 mm from the terminus of the tip (i.e. the end through which the fluid sample enters the probe).
  • the markings may, for example, be lines applied to the surface of the tip, or indentations created in the surface.
  • the present invention provides a periodontal probe comprising a detector which is configured to detect the presence and/or concentration of an analyte in a fluid in the oral cavity of a subject.
  • the detector comprises a nanopore or a nanopipette.
  • the tip of the probe (e.g. the periodontal probe) comprises or is constituted by a nanopipette.
  • the detector forms a part of, i.e. it is integrated into, the probe.
  • the tip of the probe (e.g. the periodontal probe) is hollow and houses a nanopipette.
  • a first electrode may be located inside the nanopipette, and a second electrode may be located outside of the nanopipette, inside the tip.
  • the nanopipette is a double-barrel pipette.
  • the probe or a device comprising the probe, may comprise a handle for operation of the probe.
  • a periodontal probe with an integrated analyte detector is especially beneficial.
  • the fact that dentists are already highly familiar with the use of periodontal probes means that a periodontal probe having analytical capabilities will be easily adopted.
  • a periodontal probe comprising a nanopipette will enable diseased or inflamed gum tissue to be identified with high spatial resolution (i.e. at the tooth level), and a diagnosis obtained in real time.
  • the invention thus provides a faster, more convenient and more cost effective solution to the diagnosis of periodontal disease, and accurate determination of the disease state.
  • the presence and/or concentration of one or more analytes in the fluid sample may be indicative of an oral condition or disease in a subject. In some embodiments, the presence and/or concentration of one or more analytes is indicative of the progression of the oral disease.
  • the oral disease may be periodontal disease, which may also be referred to as gum disease.
  • the periodontal disease may be gingivitis, mild periodontitis, moderate periodontitis, severe periodontitis or very severe periodontitis.
  • Gingivitis is classified according to the 2017 World Workshop classification system at a patient/case level the presence of bleeding to probe at >10% of sites in an otherwise intact periodontium, or one where any bone/attachment loss has arisen for reasons other than periodontitis.
  • Localised gingivitis is 10-30% of bleeding sites and generalised is >30% of sites.
  • a patient is a periodontitis case in the context of clinical care if:
  • Interdental clinical attachment loss is detectable at >2 non-adjacent teeth, or
  • Periodontitis is then staged and graded as: Stage 1 (mild) periodontitis is characterised by patients exhibiting ⁇ 15% or ⁇ 2 mm bone loss due to periodontitis.
  • Stage 2 (moderate) periodontitis is defined as bone loss up to the coronal third of the root, or with 3-4 mm attachment loss.
  • Stage 3 (severe) periodontitis is defined as bone loss in the mid third of the root, or with >5mm attachment loss and ⁇ 4 teeth lost.
  • Stage 4 (very severe) periodontitis is defined as bone loss in the apical third of the root, or with >5 mm attachment loss and >5 teeth lost.
  • periodontitis refers to ⁇ 30% of teeth involved and generalised is >30%.
  • progression i.e. the extent or severity of the oral disease, will therefore be understood as referring to whether the subject has gingivitis, mild periodontitis or severe periodontitis.
  • progression i.e. the extent or severity of the oral disease, will therefore be understood as referring to whether the subject has gingivitis, or mild, moderate, severe or very severe periodontitis.
  • the analyte is a biomarker.
  • One or more biomarkers may be detected to diagnose the oral disease, and/or determine the progression of the disease.
  • a combination of biomarkers may be detected.
  • the concentration of a biomarker may be increased or decreased in a fluid sample taken from a subject suffering from an oral disease, relative to the concentration present in a fluid sample taken from a subject without an oral disease, or relative to a reference or threshold value.
  • the concentration of a biomarker may be increased or decreased in a fluid sample taken from a subject without oral disease, or with an oral disease in an earlier stage of progression (e.g. gingivitis) relative to the concentration present in a fluid sample taken from a subject with an oral disease in a later stage of progression (e.g. mild, moderate, severe or very severe periodontitis).
  • the detection and/or quantification of biomarkers may be used to distinguish between a lack of oral disease, gingivitis, and different stages of periodontitis.
  • the biomarker is a protein.
  • the biomarker may be selected from the group consisting of haemoglobin alpha, haemoglobin beta, haemoglobin delta, elastase, carbonic anhydrase 1 , plastin 1 , transaldolase, S100 calcium binding protein A8 (S100-A8, also known as calgranulin A), S100 calcium binding protein A9 (S100-A9, also known as calgranulin B) or S100P, myosin-9, Alpha-1-acid glycoprotein (A1AGP), matrix metalloproteinase-9 (MM P-9), Peptidyl-prolyl cis-trans isomerase A, Haptoglobin-related protein, Alpha-N-acetylgalactosaminidase, NADPH oxidase, pyruvate kinase (PK), interleukin-1 p, free light chain kappa, free light chain lambd
  • N ⁇ dzi-Gora et al. (Cent. Eur. J Immunol. 2016; 41 (2):2) observed significantly higher concentrations of elastase and MMP-9 in patients with periodontitis compared to healthy individuals, demonstrating the utility of these proteins as biochemical indicators of the severity of periodontitis.
  • Victor et al. (J. Int. Oral Health, 2014; 6(6):67-71) found significantly elevated levels of MMP-9 among smokers with chronic periodontitis.
  • the device or system may further comprise a power supply, for example a battery or a connector for receiving mains power.
  • a power supply for example a battery or a connector for receiving mains power.
  • the system further comprises a controller (e.g. a computer).
  • the controller may be configured to control the operation of the system in use.
  • the controller may be configured to control the voltage applied to the electrodes of the nanopipette.
  • the controller may comprise a user interface.
  • the user interface may enable an operator to input instructions and/or parameters, such as voltages or timings.
  • the user interface enables the user to observe the signal generated by translocation of molecules through the nanopore.
  • the controller may comprise a memory for storing the generated signal.
  • composition comprising carrier molecules for the detection of a target analyte, each carrier molecule being functionalised with a capture moiety which is capable of specifically binding to a target analyte.
  • the target analyte may be a biomarker which is indicative of an oral disease.
  • the carrier molecules may be configured for use in the detection of target analytes using resistive pulse sensing.
  • the size and/or shape of the carrier molecules may be selected such that they are able to pass through a nanopore one by one.
  • the use of carrier molecules can facilitate the detection of target analytes using nanopores by increasing the mass of the subject molecule, thereby reducing the translocation speed and improving the signal-to-noise ratio.
  • Carrier molecules which are able to specifically bind a target analyte can be used to facilitate the detection of target analytes in a fluid using resistive pulse sensing.
  • a change in the ion current signature detected upon translocation through the nanopore, as compared to the signature of the carrier molecule alone (i.e. in the absence of the target analyte), may be indicative of the formation of a carrier molecule-target analyte complex and thus the presence of the target analyte in the sample.
  • the carrier molecule comprises at least two, at least three or at least four capture moieties. In some embodiments all of the capture moieties of a single carrier molecule are specific for the same target analyte. Alternatively, one, some or each of the capture moieties on a single carrier molecule may be specific to a different target analyte.
  • each carrier molecule may comprise a single capture moiety.
  • the carrier molecules may be functionalised with one or more capture moieties which are capable of specifically binding to any of the biomarkers identified herein.
  • the carrier molecules comprise nucleic acids.
  • the nucleic acids may be DNA, RNA, or a nucleic acid analogue, or a mixture thereof.
  • a 'nucleic acid analogue' is understood to mean a structural analogue of DNA or RNA, designed to hybridise to complementary nucleic acid sequences.
  • a nucleic acid analogue may be distinguished from DNA or RNA by its phosphate backbone, sugar groups, and/or nucleobases.
  • nucleic acid analogues include, but are not limited to, threose nucleic acid, glycol nucleic acid, morpholino oligomers, peptide nucleic acids (PNA), locked nucleic acids "LNA", 2'-O-methyl nucleic acids, 2'-fluoro nucleic acids, phosphorothioates, and metal phosphonates.
  • the carrier molecules are formed partially or entirely from nucleic acids. In some embodiments, the carrier molecules are formed from DNA. In some embodiments, the carrier molecules may be oligonucleotides.
  • the carrier molecules may be formed from single-stranded nucleic acid or double-stranded nucleic acid (e.g. single- or double-stranded DNA), or a combination thereof.
  • the carrier molecule may also comprise portions of single-stranded nucleic acid.
  • the carrier molecules comprise a double stranded backbone and at least one single-stranded portion (i.e. an overhang) extending from the backbone.
  • Oligonucleotides having a desired sequence may be synthesised chemically or using standard molecular biology techniques which are known to those skilled in the art. For example, a desired sequence may be constructed by ligating portions of the sequence into a suitable plasmid or vector. Copies of the desired sequence may then be obtained using PCR amplification.
  • carrier molecules such as via DNA self-assembly, as shown in Loh et al., 2018, Anal Chem. 90, 14063-14071.
  • Protein capture probes may be attached to a nucleic acid which forms the carrier molecules prior to assembly, using established ligation chemistry.
  • carrier molecules may be prepared using enzymatic modification of double-stranded DNA, using methods known to those skilled in the art.
  • Each carrier molecule may be no more than about 100, no more than about 50, or no more than about 20 kilo bases (for a single-stranded carrier molecule) or kilo base-pairs (for a double-stranded carrier molecule) in length. In some embodiments, each carrier molecule is no more than about 15, no more than about 10, no more than about 8, no more than about 6 or no more than about 4 kilo bases (for a single-stranded carrier molecule) or kilo base-pairs (for a double-stranded carrier molecule) in length. The longer the carrier, the more capture moieties can be incorporated. However, extremely long carrier molecules are more likely to suffer from secondary effects, such as DNA knotting or crowding in solution.
  • the capture moiety may comprise any suitable molecule which is able to selectively bind to the target analyte.
  • suitable molecules include single-stranded nucleic acids (e.g. comprising a sequence which is complementary to that of a target analyte), aptamers (nucleic acid or peptide aptamers), affimers, antibodies, proteins, molecularly imprinted polymers (MIPs) and nucleic acid-protein fusion molecules.
  • the capture moiety is an antibody.
  • antibody includes antibody fragments (e.g. Fab fragments, F(ab’)2 fragments), polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies (e.g. single chain variable fragments (scFV)).
  • the antibody may be a bispecific antibody, e.g. an antibody that has a first variable region that specifically binds to a first antigen and a second variable region that specifically binds to a second, different, antigen.
  • Antibodies may be produced using standard techniques which are well-known to the skilled person.
  • Antibody fragments may be produced by the modification of whole antibodies or synthesized de novo using known recombinant DNA methodologies.
  • each carrier molecule comprises an identifier moiety, or “barcode”.
  • the identifier moiety results in a unique signal upon translocation of the carrier molecule through a nanopore, thereby enabling carrier molecules comprising different identifier moieties to be distinguished from each other.
  • Carrier molecules which are functionalised with capture moieties that are specific for the same target analyte may each be provided with the same identifier moiety.
  • Carrier molecules functionalised with capture moieties for different target analytes may be provided with different identifier moieties. This enables several target analytes to be assayed at the same time (i.e. multiplexing).
  • An identifier moiety may be, for example, a nucleic acid structure.
  • composition may be in the form of a solution or suspension of the carrier molecules in a suitable solvent or buffer (e.g. TE buffer).
  • a suitable solvent or buffer e.g. TE buffer
  • kits for diagnosing an oral disease in a subject comprising:
  • a device comprising a probe which is configured to collect a fluid sample from the oral cavity of the subject;
  • the device and/or the probe may be one as defined herein.
  • the kit further comprises a detector which is configured to detect in the fluid sample the presence and/or concentration of an analyte which is indicative of the oral disease.
  • the kit may comprise a nanopipette.
  • the nanopipette may be provided separately from the probe.
  • the kit may comprise a nanopipette for insertion into a tip of the probe.
  • the kit comprises a plurality of nanopipettes. This enables the nanopipette to be changed between each patient or each sample.
  • the kit may further comprise a composition as defined herein.
  • the kit may additionally comprise one or more reference samples.
  • a signal generated by the reference sample be compared with a signal generated by the fluid sample obtained from the patient, thereby facilitating the detection of the presence and/or concentration of the target analyte(s), and thus a diagnosis.
  • a method of diagnosing an oral disease in a subject comprising detecting the presence and/or concentration of a target analyte in a fluid sample obtained from the oral cavity of the subject.
  • the presence or concentration of the target analyte e.g. a biomarker
  • the method may be carried out using a device, a probe or a system as defined herein.
  • the fluid sample may be obtained, or may have been previously obtained, from the oral cavity of the subject using a probe or a device as defined herein.
  • the probe is a periodontal probe having a hollow tip.
  • resistive pulse sensing is used to detect the presence and/or concentration of the target analyte.
  • the invention provides the use of resistive pulse sensing to diagnose an oral disease in a subject.
  • the method comprises using a nanopore or a nanopipette to detect the presence and/or concentration of the target analyte. Detect the presence and/or concentration of the target analyte may be carried out using a detector as defined herein.
  • the method is carried out using a device comprising a probe and an integrated detector.
  • the method may be carried out using a probe having a hollow tip in which is housed a nanopipette.
  • the integration of the probe and the detector into a single device conveniently provides a compact device for the diagnosis of oral disease.
  • the method comprises transferring the fluid sample from the probe to a separate detector which is configured to detect the presence and/or concentration of the analyte e.g. using resistive pulse sensing.
  • the detector may be as defined herein.
  • the detector may comprise a nanopore or a nanopipette.
  • the method may comprise:
  • the voltage applied across the nanopore may be from 0.05 to 10 volts, from 0.1 to 9 volts, from 0.5 to 8 volts, from 1 to 6 volts, from 2 to 5 volts or from 3 to 4 volts.
  • the method further comprises contacting the fluid sample with carrier molecules, prior to detection.
  • the carrier molecules may be functionalised with a capture moiety which is capable of specifically binding to the target analyte (e.g. a biomarker) which is indicative of the oral disease.
  • Translocation through the nanopore of a carrier molecule bound to the target analyte i.e. a carrier molecule-analyte complex
  • the fluid sample is incubated with carrier molecules prior to translocation, wherein the carrier molecules are functionalised with a capture moiety which is capable of specifically binding to a biomarker of an oral disease.
  • the fluid sample may be incubated with the carrier molecules for a period of time sufficient to enable the capture moieties to bind to the target analyte, if present in the fluid sample. Incubation may be carried out for a period of time of from 5 s to 1 hour, from 10 s to 30 minutes, from 30 s to 15 minutes, from 1 minute to 10 minutes, or from 2 minutes to 5 minutes. Incubation may be carried out at a temperature of from 10 to 35 °C, from 15 to 30 °C, from 18 to 25 °C or from 20 to 22 ° C. Suitably, incubation is carried out at room temperature (e.g. about 20 °C).
  • the signal may be a current-time signal (i.e. showing the change in the electric current across the nanopore as a function of time).
  • nanopore detection works by detecting changes to the electric current as molecules are translocated through the pore.
  • the translocation of a molecule (e.g. a carrier molecule) through the nanopore produces an event signature which is characteristic of that molecule.
  • each carrier molecule will generally produce sub-structure (sub-events) within the event.
  • the properties of these sub-events can be used to detect whether or not the carrier molecule is bound to the target analyte.
  • the signal detected may then be compared to a reference signal.
  • a reference signal By comparison to a reference signal, the presence of the target analyte in the fluid sample may be determined.
  • the reference signal may be one generated using a fluid sample obtained from a healthy subject without oral disease, or using a fluid sample which is known to contain, or known not to contain, the target analyte.
  • the reference signal is a signal generated by the translocation of unbound carrier molecules through the nanopore.
  • the reference signal may be obtained from the literature.
  • comparison of the signal detected with a reference signal may be used to determine whether the concentration of the target analyte (i.e. biomarker) in the fluid sample is elevated or reduced, relative to the reference.
  • concentration of the target analyte i.e. biomarker
  • An increase or a decrease in concentration of a particular target analyte may be indicative of disease and/or the stage of the disease.
  • detecting a concentration of the analyte includes determining a relative concentration with respect to a reference, as well as determining absolute concentration.
  • the method comprises determining, or estimating, the absolute concentration of the target analyte, using the detected signal. For example, the ratio of "bound” vs. "unbound” sub-events for a given target allows for an estimation of the target analyte concentration.
  • the absolute concentration of the target analyte present in the fluid sample may then be compared with a threshold value in order to diagnose whether the subject is suffering from an oral disease, the type of disease and/or the extent of the disease.
  • the threshold value may be one generated using a fluid sample obtained from a healthy subject without oral disease, or from a subject known to be suffering from gingivitis or periodontitis. Alternatively, the threshold value may be obtained from the literature.
  • the method comprises detecting the presence and/or concentration of a target analyte in multiple (e.g. 2, 3, 4, 5, 6 or more) fluid samples obtained from the oral cavity of the subject.
  • the fluid samples may be obtained from different sites within the oral cavity. This enables sites of inflammation or disease, or the progression of disease, to be mapped.
  • the method comprises generating a map of sites of disease within the oral cavity of the subject. The map may indicate the severity of the disease at each site.
  • the method comprises detecting the presence and/or concentration of multiple (e.g. two, three, four, five, six or more) target analytes within the fluid sample.
  • a set of biomarkers may be used to diagnose an oral disease in a subject, and/or determine the progression of the disease.
  • a panel of biomarkers may be selected for distinguishing between a healthy subject (i.e. lack of oral disease) and a subject with inflammation or gingivitis, between gingivitis and periodontitis, or for distinguishing between different states of periodontitis (e.g. between mild and moderate, or moderate and severe periodontitis).
  • a method of diagnosing an oral disease in a subject comprising: - transferring a fluid sample from a probe to a detector, wherein the fluid sample was previously collected from the oral cavity of the subject using the probe; and
  • the detector detecting in the fluid sample the presence and/or concentration of a target analyte which is indicative of the oral disease.
  • the invention provides a method for detecting the presence and/or concentration of a target analyte in a fluid sample using resistive pulse sensing, wherein the target analyte is a biomarker of an oral disease.
  • the fluid sample may be one which was previously obtained from the oral cavity of a subject.
  • the method further comprises obtaining the fluid sample from the oral cavity of the subject.
  • the fluid sample may be obtained by any convenient means, for example using electroosmosis, suction or capillary action.
  • the fluid sample is obtained using a probe as defined herein.
  • the probe comprises a periodontal probe housing a nanopipette
  • a new nanopipette may be provided for each fluid sample obtained (e.g. for each tooth or each GCF measurement. This will avoid cross-contamination between teeth and allow for sterilisation of the periodontal probe (in the absence of the nanopipette) between samples.
  • the methods of the invention may be used to determine whether a subject is suffering from gingivitis, mild periodontitis or severe periodontitis.
  • the method further comprises treating a subject diagnosed as having an oral disease.
  • T reatment may include one or more of: scaling; root planning; administration of antibiotics (e.g. oral or topical); surgery; regular dental cleaning (e.g. every 3 or 6 months); and use of mouthwash (e.g. daily).
  • the fluid sample may be saliva or gingival crevicular fluid (GCF).
  • GCF gingival crevicular fluid
  • the subject may be a human or a non-human mammal, such as a horse, cow, pig, goat, sheep, dog, cat or primate.
  • the invention also relates to a modular polynucleotide and to a method of making a modular polynucleotide.
  • the invention also relates to a method and a kit for determining if one or more analyte(s) is/are present in a sample, and a method and kit for diagnosis of a medical condition, using a modular polynucleotide.
  • the invention also extends to a method of treatment, and a method of determining the efficacy of a therapeutic agent.
  • Assays are an important aspect of investigative research as they enable one to measure the presence, amount and/or functional activity of an analyte.
  • Each analyte of interest may require a different specialised technique or equipment to detect its presence.
  • a sample suspected of containing several different analytes may require the use of gas chromatographymass spectrometry (GC-MS) or high pressure liquid chromatography -mass spectrometry (HPLC-MS) to detect analytes present at low concentrations; enzyme linked immunosorbent assays (ELISA) or immunofluorescence (IF) to detect analytes in the form of polypeptides or proteins; and polymerase chain reaction (PCR) or a microarray to detect nucleic acids.
  • GC-MS gas chromatographymass spectrometry
  • HPLC-MS high pressure liquid chromatography -mass spectrometry
  • ELISA enzyme linked immunosorbent assays
  • IF immunofluorescence
  • PCR polymerase chain reaction
  • the inventors have surprisingly developed a novel method of creating a modular doublestranded polynucleotide having subunits (i.e. (im)mature conjugate subunits) each comprising a probe for binding an analyte.
  • the modular polynucleotide can be used with a carrier- enhanced resistive pulse sensing technology (e.g. nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing) to simultaneously detect several different analytes, including different types of analyte, in a high- throughput manner.
  • a carrier- enhanced resistive pulse sensing technology e.g. nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing
  • a method of making an immature conjugate subunit comprising: a) conjugating a (first) probe, for binding a (first) analyte, to a probe conjugation site of a (first) double-stranded polynucleotide, to create a (first) immature conjugate subunit.
  • a method of making a mature conjugate subunit comprising: a) conjugating a (first) probe, for binding a (first) analyte, to a probe conjugation site of a (first) double-stranded polynucleotide, to create a (first) immature conjugate subunit; and b) forming a (first) mature conjugate subunit by cleaving the double-stranded polynucleotide of the (first) immature conjugate subunit to form a first sticky end, or a first sticky end and/or a second sticky end.
  • the first sticky end and/or the second sticky end of the mature conjugate subunit may be complementary to a (first or second) sticky end of a further, separate mature conjugate subunit, or complementary to a (first or second) sticky end of a further, separate mature conjugate subunit to be formed from an immature conjugate subunit.
  • Each mature conjugate subunit may further comprise a double-stranded polynucleotide spacer.
  • the spacer may be annealed to the double-stranded polynucleotide of the (first) mature conjugate subunit so as to create a polynucleotide backbone.
  • the method according to the sixth aspect may further comprise the step of: c) annealing the first sticky end or the second sticky end of the (first) mature conjugate subunit to a complementary sticky end of a (first) double-stranded polynucleotide spacer.
  • the invention may further comprise creating a modular polynucleotide comprising a doublestranded polynucleotide backbone having two or more mature conjugate subunits.
  • the method according to the sixth aspect may further comprise:
  • the method of creating a modular polynucleotide may comprise performing a one-pot synthesis reaction.
  • the cleaving and annealing steps may be performed simultaneously in a single reaction vessel.
  • the modular polynucleotide may be created by placing multiple immature conjugate subunits in a single reaction vessel together with an exonuclease, a DNA ligase and a DNA polymerase, and optionally multiple double-stranded polynucleotide spacers.
  • the modular polynucleotide may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1000 or more, 3000 or more, 5000 or more, 10000 or more mature conjugate subunits.
  • the modular polynucleotide may comprise between two and 10000 mature conjugate subunits, two and 5000 mature conjugate subunits, two and 1000 mature conjugate subunits, two and 500 mature conjugate subunits, two and 100 mature conjugate subunits, two and 50 mature conjugate subunits, or two and 10 mature conjugate subunits.
  • the method of the invention may ultimately comprise attaching a plurality of mature conjugate subunits (each comprising a probe) together in a modular fashion.
  • the initial polynucleotide used to create the mature conjugate subunits may be a blunt-ended polynucleotide.
  • Prior art methods of attaching blunt-ended polynucleotides together can only be achieved with polynucleotides that are at least 100 base pairs in length (and by using an exonuclease, a DNA polymerase and a DNA ligase).
  • the method according to the invention can be used to attach several different blunt-ended polynucleotides together that are significantly shorter in length (e.g. blunt-ended polynucleotides that are between about 20 and about 100 base pairs in length or between about 34 and about 100 base pairs in length).
  • nucleases e.g. exonucleases
  • nucleases such as exonucleases, e.g. T5 exonuclease
  • nucleases are unable to navigate across the site at which the steric hindering agent is located (e.g. the probe), and thus get knocked off of the blunt-ended polynucleotide.
  • the method according to the invention enables the blunt end of a short double- stranded polynucleotide to be converted into “sticky ends” without converting the entire double-stranded polynucleotide into a single-stranded polynucleotide.
  • the method according to the invention is also advantageous because it enables probes for different analytes, different classes of analyte and the same analyte to be conjugated to a single polynucleotide backbone of a modular polynucleotide. Consequently, a modular polynucleotide according to the inventio can be used to perform multiplex, high-throughput analysis using a single technique (i.e. carrier- enhanced resistive pulse sensing, such as nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing).
  • carrier- enhanced resistive pulse sensing such as nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing.
  • the method according to the invention enables each probe of a modular polynucleotide to be conjugated at a specific location within the polynucleotide backbone. Consequently, each disruption of the electrical current generated during use with a carrier- enhanced resistive pulse sensing technology can be attributed to a particular probe, and the specific characteristics of each disruption can be used to determine if a probe has bound an analyte or not. Furthermore, the method can be used to easily modify a modular polynucleotide so as to alter the analyte(s) being detected due to the modular nature of the immature conjugate subunits, which may each comprise an optional double-stranded polynucleotide spacer.
  • the probe conjugation site may comprise a nucleotide sequence specific for a transferase enzyme.
  • the probe conjugation site may comprise or consist of a CpG island (or a CpG thereof) or the nucleotide sequence TCGA.
  • the probe conjugation site comprises a nucleotide sequence specific for a methyl transferase enzyme (e.g. m.Taql).
  • the probe conjugation site may comprise the nucleotide sequence TCGA, or a CpG island (or a CpG thereof).
  • a double-stranded (im)mature conjugate subunit may be created by a method according to the invention.
  • an immature conjugate subunit comprising: a probe, for binding an analyte, conjugated to a probe conjugation site of a double-stranded polynucleotide, wherein the probe conjugation site comprises a nucleotide sequence specific for a transferase enzyme.
  • the polynucleotide of the double-stranded mature conjugate subunit may be between about 20 and about 100 base pairs/nucleotides in length, or between about 34 and about 100 base pairs/nucleotides in length.
  • the polynucleotide of the double-stranded mature conjugate subunit may comprise blunt ends (e.g. a first blunt end and/or a second blunt end).
  • the polynucleotide of the double-stranded mature conjugate subunit may comprise sticky ends (e.g. 5’-sticky ends or 3’-sticky ends).
  • the conjugate subunit may be referred to as a mature conjugate subunit.
  • a double-stranded modular polynucleotide may be created by a method according to the invention.
  • a modular polynucleotide may be created by annealing a plurality of mature conjugate subunits together.
  • Each of the mature conjugate subunits may be created by a method according to the invention.
  • a double-stranded modular polynucleotide comprising: a double-stranded polynucleotide backbone comprising a plurality of probe conjugation sites each site having a nucleotide sequence specific for a transferase enzyme, wherein each probe conjugation site is separated by at least 16 base pairs, and wherein at least one probe conjugation site is conjugated to a single probe for binding an analyte.
  • At least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the sites may be conjugated to a probe. 100% of the probe conjugation sites may be conjugated to a probe.
  • the modular polynucleotide may comprise different classes of probe, which are capable of binding to the same analyte or different analytes.
  • the modular polynucleotide may comprise two or more different classes of probe, which are capable of binding to the same analyte or a different analyte.
  • the (im)mature conjugate subunit according to the seventh aspect may be used to form a modular polynucleotide according to the eighth of invention or a modular polynucleotide made by a method according to the invention.
  • a single modular polynucleotide according to the invention may be used to perform multiplex, high- throughput analysis using a carrier- enhanced resistive pulse sensing technology to simultaneously detect different analytes using different probes.
  • the method according to the invention is also advantageous because it can be used to create a modular polynucleotide comprising a plurality of identical probes. None, some or all of the probes of such a modular polynucleotide may be capable of binding to relevant analyte molecules in a sample. Thus, such a modular polynucleotide can be used to improve the signal-to-noise ratio and/or reduce the total analysis time. The modular polynucleotide can also be used to provide quantitative information about analyte binding (analyte concentration in a sample).
  • the probe conjugation site referred to herein may comprise a nucleotide sequence specific for a transferase enzyme (e.g. a methyl transferase enzyme).
  • the probe conjugation site may comprise or consist of a CpG island (or a CpG thereof) or a “TCGA” nucleotide sequence as they are specific for methyl transferases.
  • the probe conjugation site comprises a nucleotide sequence specific for a methyl transferase enzyme (e.g. m.Taql).
  • the probe conjugation site may comprise the nucleotide sequence TCGA.
  • the methyl transferase enzyme may be an enzyme that uses S-adenosyl methionine (SAM) as a starting reagent.
  • SAM S-adenosyl methionine
  • the methyl transferase enzyme may be an enzyme that binds SAM.
  • Each of the probe conjugation sites may be separated by at least about 15 base pairs, at least about 30 base pairs, at least about 100 base pairs, or at least about 300 base pairs. The greater the distance between each probe conjugation site, the easier it is to detect a conjugated probe or an analyte bound to a conjugate probe. Most preferably each of the probe conjugation sites is separated by between about 300 base pairs and about 1000 base pairs.
  • a modular polynucleotide according to the invention can be created (see Examples 1 and 2). It is a simple and robust solution to investigate a diverse range of analytes (e.g. biomarkers) quickly, simultaneously and at a low cost.
  • Modular polynucleotides can be flexibly designed to carry a range of different probes, or multiple identical probes, depending on the application.
  • the invention provides a novel way of creating modular polynucleotides with improved efficiency, in terms of time, cost and design flexibility.
  • conjugation of one or more probes to a polynucleotide may be determined by a carrier-enhanced resistive pulse sensing technology (see Loh et al., Anal. Chem. 2018, 90 (23): 14063-14071).
  • a carrier-enhanced resistive pulse sensing technology see Loh et al., Anal. Chem. 2018, 90 (23): 14063-14071).
  • modular polynucleotides and (im)mature conjugate subunits according to the invention may be used with a carrier- enhanced resistive pulse sensing technology to detect the binding of analytes in a sample.
  • a method of creating a recombinant polynucleotide comprising:
  • the method according to the invention is advantageous because it would enable the creation of specific sequences that can bridge two or more double-stranded polynucleotides together without the use of a specific restriction site or a large insert. Consequently, less “junk” DNA and more coding DNA can be inserted into viral vectors. Viral vectors have a limited capacity for inserts DNA.
  • a method of creating sticky ends on a blunt-ended polynucleotide comprising: a) (i) conjugating a steric hindering agent to a conjugation site of a polynucleotide comprising a first blunt end and/or a second blunt end, or
  • the method may optionally further comprise: b) cleaving the first blunt end and/or the second blunt end of the polynucleotide using a nuclease to create a first sticky end and/or a second sticky end.
  • the steric hindering agent may be a probe as defined herein.
  • the steric hindering agent impedes the activity of nucleases (e.g. exonucleases) that may be used to cleave short (i.e. less than 100 base pairs) blunt-ended double-stranded polynucleotides (e.g. an immature conjugate subunit).
  • nucleases e.g. exonucleases
  • blunt-ended double-stranded polynucleotides e.g. an immature conjugate subunit
  • the method according to the invention enables the blunt ends of a short double-stranded polynucleotide to be converted into “sticky ends” without converting the entire double-stranded polynucleotide into a single-stranded polynucleotide.
  • Each probe may be conjugated to a separate polynucleotide using any method known in the art.
  • the conjugating step may comprise chemically-modifying or functionalising the polynucleotide with an adaptor to facilitate conjugation of the probe to the polynucleotide.
  • the conjugating step may comprise chemically- modifying or functionalising the probe conjugation site (e.g. TCGA or a CpG of a CpG island) of the double-stranded polynucleotide with an adaptor.
  • the probe conjugation site e.g. TCGA or a CpG of a CpG island
  • One or more, two or more, three or more, or four more nucleotides of the probe conjugation site may be chemically modified.
  • the conjugating step may comprise chemically- modifying or functionalising the probe conjugation site of the polynucleotide with an azide (N3) group or a peptide adaptor.
  • the conjugating step comprises chemically attaching an azide group to the probe conjugation site.
  • the nucleotides of the probe conjugation site are not terminal base-paired nucleotides (i.e. nucleotides located within four nucleotides of the 5’-basepaired terminus or the 3’- basepaired terminus of the polynucleotide).
  • the probe conjugation site is at least 15 nucleotides from the 5’-terminus and the 3’-terminus of the polynucleotide.
  • the conjugating step may comprise attaching an azide group to the polynucleotide by contacting the polynucleotide with an azide donor.
  • the azide donor may be a substance comprising an azide group and that has the ability to donate the azide group without reacting to other groups on the recipient molecule.
  • the azide donor may be RAdoHcy-8-Hy -PEG-N3, a modified SAM molecule containing an azide group (rather than a methyl) or an azide modified nucleotide.
  • the azide donor is RAdoHcy-8-Hy -PEG-N3 or a modified SAM molecule containing an azide group.
  • the conjugating step may comprise chemically-modifying or functionalising the polynucleotide with an azide (-N3) group by contacting the polynucleotide with an azide donor (e.g. RAdoHcy-8-Hy-PEG-N3 or a SAM molecule containing an azide group) in the presence of a catalyst, such as an enzyme.
  • the enzyme may be a methyl transferase.
  • the methyl transferase is Taql methyl transferase (EC2.1.1.72).
  • the conjugating step may comprise chemically-modifying or functionalising the polynucleotide with an azide (N3) group by contacting the polynucleotide with an azide donor (e.g.
  • RAdoHcy-8- Hy-PEG-N3 or a SAM molecule containing an azide group in the presence of a methyl transferase, such as Taql methyl transferase (EC2.1.1.72) for about 30 minutes to about 90 minutes at about 50oC.
  • a methyl transferase such as Taql methyl transferase (EC2.1.1.72)
  • the conjugating step may comprise chemically-modifying or functionalising the probe to facilitate conjugation to the polynucleotide without interfering with the ability of the probe to bind an analyte.
  • the probe should be functionalised in a region that is not responsible for binding to an analyte.
  • the probe is an antibody
  • the antibody should not be functionalised in or near the antibody binding region (e.g. Fab).
  • the antibody should be functionalised in a region such as the Fc region.
  • the terminus of Fc region e.g. CH3 or CH2
  • the linker is functionalised.
  • the 5’end of the single stranded DNA/RNA is functionalised with an amine group for conjugation.
  • the 3’end is functionalised.
  • the conjugating step may comprise conjugating the probe directly or indirectly to the polynucleotide.
  • the conjugating step may comprise covalently conjugating the probe directly to the polynucleotide.
  • the conjugating step may comprise conjugating the probe to the polynucleotide via a photocleavable bond (e.g. a UV-sensitive bond).
  • the conjugating step may comprise conjugating (e.g. covalently conjugating) the probe directly or indirectly to the polynucleotide conjugation site.
  • the probe is not conjugated to a terminal basepaired nucleotide.
  • the probe conjugation site is not located within 15 base pairs from the 5’-terminus or the 3’-terminus of the polynucleotide.
  • the probe may be conjugated to a polynucleotide that has been chemically-modified or functionalised.
  • the probe may be conjugated to a functional group or chemical group of a nucleotide that has been chemically- modified or functionalised.
  • the conjugating step may comprise conjugating the probe to the double-stranded polynucleotide via a linker (e.g. DBCO-NHS ester, a peptide nucleic acid, a histidine tag).
  • the conjugating step may comprise covalently conjugating the probe to the double-stranded polynucleotide via a linker (e.g. DBCO-NHS ester, a peptide nucleic acid or a histidine tag).
  • the conjugating step may comprise chemically-modifying or functionalising the probe with a DBCO-NHS ester, a peptide nucleic acid or a histidine tag.
  • the conjugating step comprises chemically-modifying or functionalising the probe with a DBCO-NHS ester.
  • the conjugating step may comprise chemically-modifying or functionalising a terminal monomer of the probe with a DBCO-NHS ester.
  • the conjugating step may comprise chemically-modifying or functionalising a terminal monomer with an amine group.
  • the conjugating step may comprise chemically-modifying or functionalising a terminal amine group of the probe with a DBCO-NHS ester.
  • the conjugating step may comprise chemically-modifying or functionalising the probe with an amine group, followed by functionalising the amine group with a DBCO-NHS ester.
  • the skilled person would appreciate how to functionalise a variety of different types of probes with an amine group, particularly the terminal monomer of the probe.
  • the conjugating step may comprise chemically-modifying or functionalising the probe by contacting it with a DBCO-NHS ester.
  • the conjugating step may comprise chemically-modifying or functionalising a terminal amine group of the probe by contacting it with a DBCO-NHS ester.
  • the probe or amine group of the probe may be contact with the DBCO-NHS ester at group at about 18°C to 25°C for about 2 hours to about 16 hours.
  • the conjugating step may comprise contacting a functionalised polynucleotide with a functionalised probe.
  • the conjugating step comprises contacting a polynucleotide functionalised with a peptide adaptor with a probe functionalised with a PNA or a histidine tag (e.g. a histidine-tagged antibody), in order to conjugate the probe to the polynucleotide.
  • the conjugating step comprises contacting a polynucleotide functionalised or chemically modified with an azide group with a probe functionalised with a DBCO-NHS ester, in order to conjugate the probe to the polynucleotide.
  • the probe conjugation site of the polynucleotide may be functionalised with an azide group.
  • the conjugating step may comprise contacting a polynucleotide functionalised with an azide group with a functionalised probe in an azide-alkyne reaction, preferably a copper-free azidealkyne reaction.
  • the polynucleotide functionalised with an azide group may be contacted with a probe functionalised with a DBCO-NHS ester at about 18°C to 25°C for about 1 hour to about 2 hours, or at about 2°C to about 6°C for about 16 hours to 24 hours.
  • the polynucleotide functionalized with an azide group is contacted with a probe functionalised with a DBCO-NHS ester at about 18°C to 25°C for about 1.5 hours, or at about 4°C for about 16 hour to 24 hours.
  • the conjugating step may comprise conjugating the probe to a probe conjugation site of the double-stranded polynucleotide via a photocleavable bond (e.g. a UV-sensitive bond).
  • a photocleavable bond e.g. a UV-sensitive bond
  • the double-stranded polynucleotide of the (im)mature conjugate subunit may be a polynucleotide comprising artificial nucleotides or natural polynucleotides.
  • One or more of the nucleotides may comprise an epigenetic modification, such as a methylation.
  • the doublestranded polynucleotide may be DNA.
  • the polynucleotide may be DNA comprising a probe conjugation site.
  • the probe conjugation site may comprise or consist of a nucleotide sequence specific for a transferase enzyme (e.g. the nucleotides TCGA, or a CpG of a CpG island).
  • the probe conjugation site may be positioned at least 8 base pairs/nucleotides from the 5’- terminal nucleotide/base pair and at least 8 base pairs/nucleotides from the 3’- terminal nucleotide/base pair.
  • the probe conjugation site may be positioned at least about 15 nucleotides from the 5’-terminus and/or the 3’-terminus; at least about 20 nucleotides from the 5’-terminus and/or the 3’-terminus; at least about 25 nucleotides from the 5’-terminus and/or the 3’-terminus; at least about 30 nucleotides from the 5’- terminus and/or the 3’- terminus; at least about 35 nucleotides from the 5’-terminus and/or the 3’-terminus; at least about 40 nucleotides from the 5’-terminus and/or the 3’-terminus; or at least about 45 nucleotides from the 5’-terminus and/or the 3’-
  • the probe conjugation site sequence may be positioned between about 15 nucleotides and about 48 nucleotides from the 5’- terminus and the 3’-terminus. Preferably the probe conjugation site sequence is positioned about 30 nucleotides from the 5’-terminus and at least 30 nucleotides from the 3’-terminus.
  • the double-stranded polynucleotide may be between about 20 and about 100 base pairs/nucleotides in length or between about 34 and about 100 base pairs/nucleotides in length. Preferably the double-stranded polynucleotide is about 60 base pairs/nucleotides in length.
  • the double-stranded polynucleotide of the immature conjugate subunit may comprise blunt ends (e.g. a first blunt end and/or a second blunt end).
  • the double-stranded polynucleotide of the mature conjugate subunit may comprise sticky ends (e.g. 5’- sticky ends or 3’-sticky ends).
  • the probe may be any agent that selectively or specifically binds to an analyte.
  • the probe may bind selectively or specifically to an analyte.
  • Preferably the probe binds specifically to an analyte.
  • Each or two or more of the probes of the modular polynucleotide may be capable of binding to the same analyte or may be identical.
  • Each or two or more of the probes of the modular polynucleotide may be capable of binding to a different analyte.
  • the probe may be any probe known in the art.
  • the probe may be a polymer (e.g. a ssRNA, a morpholino or a peptide nucleic acid).
  • a polymer e.g. a ssRNA, a morpholino or a peptide nucleic acid.
  • the probe may be one or more members selected from the group comprising a polypeptide, a protein (e.g. an antibody or an affimer), a polynucleotide (e.g. DNA or single-stranded DNA), a nanoparticle and an aptamer.
  • the probe(s) is/are one or more selected from the group consisting of a singlestranded nucleotide, an antibody, a (functional) fragment of an antibody and an aptamer.
  • the probe may be DNA, preferably single-stranded DNA, or RNA, preferably single- stranded DNA.
  • the probe may be an antibody or a (functional) fragment thereof (e.g. a scFv, a VL, a VH, a Fd; an Fv, an Fab, a Fab', a F(ab')2, an Fc fragment, or a bispecific antibody) that binds to an analyte.
  • a scFv a VL, a VH, a Fd
  • an Fv an Fab, a Fab', a F(ab')2, an Fc fragment, or a bispecific antibody
  • the probe may be an antibody that binds to the sepsis biomarkers Interleukin 6 (IL-6) or Procalcitonin.
  • IL-6 Interleukin 6
  • Procalcitonin Procalcitonin
  • antigen-binding region can mean a region of the antibody having specific binding affinity for its target antigen/analyte.
  • the binding region may be a hypervariable CDR or a functional portion thereof.
  • functional portion of a CDR can mean a sequence within the CDR which shows specific affinity for the target analyte.
  • (functional) fragment" of an antibody can mean a portion of the antibody which retains a functional activity.
  • a functional activity can be, for example antigen binding activity or specificity.
  • VL fragment can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs.
  • a VL fragment can further include light chain constant region sequences.
  • VH fragment can means a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.
  • Fd fragment can mean the heavy chain variable region coupled to the first heavy chain constant region, i.e. VH and CH-i.
  • the "Fd fragment” does not include the light chain, or the second and third constant regions of the heavy chain.
  • Fv fragment can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains.
  • the variable regions of the heavy and light chains include, for example, the CDRs.
  • an Fv fragment includes all or part of the amino terminal variable region of about no amino acids of both the heavy and light chains.
  • Fab fragment can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment.
  • a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains.
  • Fab 1 fragment can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment.
  • a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain.
  • a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.
  • the antibody fragment may alternatively comprise a Fab'2 fragment comprising the hinge portion of an antibody.
  • F(ab) fragment can mean a bivalent antigen-binding fragment of a human monoclonal antibody.
  • An F(ab) fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.
  • single chain Fv (scFv) can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.
  • bispecific antibody can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide.
  • CDR can mean a hypervariable region in the heavy and light variable chains. There may be one, two, three or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e. the three-dimensional combining site with which the antigen binds or specifically reacts. It has however been postulated that there may be four CDRs in the heavy chains of some antibodies.
  • CDR also includes overlapping or subsets of amino acid residues when compared against each other.
  • residue numbers which encompass a particular CDR or a functional portion thereof will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.
  • the mature conjugate subunit may be created by cleaving the double-stranded polynucleotide of the immature conjugate subunit with an enzyme.
  • the enzyme may be a nuclease enzyme. Most preferably the enzyme is an exonuclease. Exonucleases are enzymes that cleave terminal 3’ and/or 5’ nucleotides from a polynucleotide, such as a blunt-ended polynucleotide, to create sticky ends.
  • the enzyme may be a 5’ exonuclease or a 3’ exonuclease.
  • the enzyme is a 5’ exonuclease, such as a T5 exonuclease.
  • a sticky end refers to a series of unpaired nucleotides in a double stranded oligonucleotide.
  • Sticky ends are created by nucleases (e.g. endonucleases or exonucleases) creating a staggered cut in a double-stranded polynucleotide.
  • Nucleases typically have recognition sequences that vary in the number of nucleotides from 4 to 6 and even 8. The longer the sticky ends, the higher the melting temperature of the double-stranded polynucleotide and the higher the number of potential sequences for recognition. Also, the longer the sticky ends, the greater the possibility of a single-strand break (DNA base hydrolysis).
  • the sticky ends of the nucleotides referred to herein may be at least about 5, at least or about 6 or at least or about 7 nucleotides in length.
  • the sticky ends of the nucleotides are about 15 to about 25 nucleotides in length.
  • the sticky ends of the nucleotide are about 20 nucleotides in length.
  • Sticky ends are preferably 20 nucleotides in length because it provides 160,000 potential sequences for recognition. Sticky ends that are about 20 nucleotides in length are also preferred because they provide a moderately high melting temperature, thus preventing separation of the strands during enzymatic construction of the modular polynucleotide.
  • the length of the sticky ends is dependent on the nuclease used to cleave the polynucleotide.
  • the nuclease may be an exonuclease, preferably a T5 exonuclease. T5 exonucleases create sticky ends that are about 20 nucleotides in length.
  • the enzyme used to create sticky ends in a doublestranded polynucleotide can also be used to cleave a separate double-stranded polynucleotide so as to make the sticky ends of both polynucleotides complementary.
  • the double-stranded polynucleotide of an immature conjugate subunit and a double-stranded polynucleotide spacer may be cleaved by th same nuclease (such as an exonuclease, e.g. T5 exonuclease) so as to create complementary sticky ends that may be used to anneal the spacer and subunit together to form a modular polynucleotide.
  • the modular polynucleotide may be created by one-pot synthesis (mixing all of the reactants in a single reaction vessel as opposed to making the modular polynucleotide in a stepwise fashion).
  • one-pot synthesis can be used to create a modular polynucleotide according to the invention due to the polynucleotide of each immature conjugate subunit comprising a nucleotide sequence that once cleaved will be complementary to a separate mature conjugate subunit.
  • each polynucleotide i.e.
  • the order of the probes in a modular polynucleotide may be arranged at user’s discretion due to the modular nature of the mature conjugate subunits.
  • the sticky ends referred to herein may be 5’ sticky ends or 3’ sticky ends.
  • the spacer may be a double-stranded polynucleotide.
  • the spacer referred to herein may be a double-stranded polynucleotide comprising a nucleotide sequence that once cleaved (e.g. with an exonuclease, such as T5 exonuclease) will comprise one or two sticky ends that are complementary to the sticky ends of a mature conjugate subunit or will be complementary to the sticky ends of a mature conjugate subunit once it has been formed.
  • the spacer may comprise artificial nucleotides or natural polynucleotides.
  • the double-stranded polynucleotide is DNA.
  • One or more of the nucleotides may comprise an epigenetic modification, such as a methylation.
  • the double-stranded polynucleotide spacer (e.g. DNA) may be at least about 100 or more, 200 or more, 300 or more, 500 or more, 1000 or more base pairs in length.
  • the doublestranded polynucleotide spacer (e.g. DNA) may be between about 300 and 2000 base pairs in length or between about 500 base pairs and about 1000 base pairs in length.
  • the presence of the spacer within the modular polynucleotide enables the distance between each probe to be controlled so as to increase the resolution of each signal produced by each probe but also to prevent each probe from being too spaced apart. Furthermore, the spacer can be used to control the order of the probes in the modular polynucleotide according to the invention.
  • the annealing step may comprise using any method known in the art to anneal complementary sticky ends together.
  • the annealing step may comprise contacting a first mature conjugate subunit with a second or several other mature conjugate subunits.
  • the annealing step may comprise contacting a mature conjugate subunit with a double-stranded polynucleotide spacer.
  • the annealing step may comprise an enzyme.
  • the annealing step occurs in the presence of an enzyme.
  • the enzyme may be a DNA ligase and/or a DNA polymerase.
  • DNA ligase joins or catalyses the joining of complementary sticky ends together.
  • DNA polymerase inserts nucleotides into gaps of an incompletely synthesised/annealed double-stranded polynucleotide, such as modular polynucleotide referred to herein.
  • Preferably contacting a mature conjugate subunit with a double-stranded polynucleotide spacer occurs in the presence of a DNA ligase and a DNA polymerase.
  • the DNA ligase may be Taq ligase.
  • the DNA polymerase may be Taq DNA polymerase.
  • Contacting a mature conjugate subunit with a double-stranded polynucleotide spacer may occur in the presence of a DNA ligase and a DNA polymerase for about 1 hour to 18 hours at about 40°C to about 50°C. Performing the annealing step under these conditions ensures that only complementary sticky ends anneal to each other (i.e. prevents mismatching of sticky ends). Mismatched base pairs are unstable at temperatures between about 40° C to about 50°C.
  • the modular polynucleotide may comprise two or more different probes that are capable of binding to the same analyte or a different analyte.
  • the modular polynucleotide may comprise two or more different classes of probe that are capable of binding to the same analyte or a different analyte.
  • the modular polynucleotide may comprise two or
  • the modular polynucleotides and mature conjugate subunits of the invention may be used for a variety of purposes, including for example, disease diagnosis, the food industry (e.g. testing food and water quality), waste analysis (e.g. nuclear and industrial waste analysis), environmental analysis (e.g. soil and atmosphere aspirational analysis).
  • the food industry e.g. testing food and water quality
  • waste analysis e.g. nuclear and industrial waste analysis
  • environmental analysis e.g. soil and atmosphere aspirational analysis
  • the inventors have developed a kit comprising components that can be used with a carrier- enhanced resistive pulse technology (e.g. nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing technology) to determine if one or more analytes is present in a sample.
  • a carrier- enhanced resistive pulse technology e.g. nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing technology
  • kits for determining if one or more analyte(s ) is/are present in a test sample comprising:
  • kits for diagnosing a test subject suffering from a medical condition comprising:
  • a modular polynucleotide according to the invention or a modular polynucleotide made by a method according to the invention, wherein the presence of one or more analyte(s) in a bodily sample from a test subject is indicative that the subject suffers from the medical condition, or wherein the absence of the one or more analyte(s) from a bodily sample from a test subject is indicative that the subject suffers from the medical condition.
  • a kit according to the ninth aspect may be used to make a modular polynucleotide according to the invention.
  • a modular polynucleotide according to the invention may be used to perform multiplex, high-throughput analysis using a carrier-enhanced resistive pulse sensing technology for the detection of different analytes using different probes.
  • the kit further comprises at least one control or reference sample.
  • the kit may comprise a control (e.g. a negative control and/or a positive control).
  • the negative control may be a sample that does not comprise one or more of the analyte(s) to be detected.
  • the positive control may be a sample that comprises one or more of the analyte(s) to be detected.
  • the kit may comprise a buffer for the samples.
  • the kit may comprise one or more enzymes selected from the group consisting of a DNA polymerase, a nuclease (e.g. exonuclease) and a DNA ligase.
  • the tenth aspect provides a method of determining if one or more analyte(s) is/are present in a test sample, the method comprising: i. contacting a modular polynucleotide according to the invention or a modular polynucleotide made by a method according to the invention with a test sample; and then ii. analysing the modular polynucleotide using a carrier enhanced-resistance pulse sensing technology to determine if one or more analyte(s) is/are present in the test sample.
  • the test sample may be an environmental sample (e.g. a soil sample or atmospheric sample), a food industry, a water sample, a waste sample (e.g. nuclear or industrial waste sample) or a bodily sample that has been taken from a test subject.
  • the method according to the invention may not comprise taking a sample from a test subject or performing surgery on a test subject.
  • the method according to the tenth aspect may be used to diagnose if a test subject suffers from a medical condition or disease.
  • the eleventh aspect provides a method of diagnosing a test subject with a medical condition, the method comprising: i. contacting a modular polynucleotide according to the invention or a modular polynucleotide made by a method according to the invention with a bodily sample taken from the test subject; and then ii.
  • the method according to the eleventh aspect is advantageous because it can be used to detect several different types of biomarkers simultaneously. Thus, the method provides a faster way to detect several different analytes and can be used to provide a more accurate diagnosis than known methods.
  • the method of the eleventh aspect may comprise administering a therapeutic agent that treats the medical condition or disease to a subject.
  • a method of treating a subject suffering from a medical condition comprising: diagnosing a test subject with a medical condition using a method according to the invention; and administering a therapeutically effective amount of a therapeutic agent for treating the medical condition.
  • a therapeutic agent for use in treating a medical condition in a subject diagnosed with a medical condition using a method according to the invention.
  • a method of determining the efficacy of a therapeutic agent being used to treat a subject’s medical condition comprising: i. diagnosing a test subject with a medical condition using a method according to the invention; ii. administering a therapeutically effective amount of a therapeutic agent for treating the medical condition; iii. contacting a modular polynucleotide- according to the invention or a modular polynucleotide made by a method according to the invention with a test sample; and iv.
  • Resistive pulse sensing technology requires two solutions to be separated by a narrow channel. A voltage is applied across the channel and (charged) molecules from one solution will move through the channel in the direction of the electric field. As they cross through the channel, the current passing between the electrodes will change. The change in current, and the duration of the change are directionally proportional to the widest diameter, and dimensions of the molecule passing through (see Figure 8).
  • Examples of carrier-enhanced resistive pulse sensing technology include nanopore- based resistive pulse sensing, such as a biological nanopore (e.g. a Phi29 Connector channel), and nanopipette-based resistive pulse sensing technology.
  • the analyte is an agent that is capable of being bound by a probe.
  • the analyte may be any substance present in a test sample (e.g. a biological or bodily sample or a non- biological sample or an environmental sample, a waste sample, a food sample or a water sample).
  • a test sample e.g. a biological or bodily sample or a non- biological sample or an environmental sample, a waste sample, a food sample or a water sample.
  • the analyte may be a biological agent or a chemical agent.
  • the sample may be a fluid, such as a liquid or a gas.
  • the sample may be a gas that has been condensed into a liquid.
  • the sample is a liquid.
  • the analyte is at least 2 nm in length.
  • the sample may be a biological sample, such as a biological liquid.
  • the analyte can be an analyte that is secreted from cells.
  • the analyte can be an analyte that is present inside cells such that the analyte must be extracted from the cells before the invention can be carried out.
  • the analyte can be an analyte that is present in a sample of fluid in which the biological organism is located.
  • the sample may be in vitro, in vivo or ex vivo.
  • the invention may be carried out in vitro on a sample obtained from or extracted from a biological organism.
  • the biological organism or test subject may be a bacterium, a protista, a fungi, a plant or an animal.
  • the biological organism may be a mammal, such as a human.
  • the sample may be a bodily sample, such as a mammalian bodily sample, e.g. a human bodily sample.
  • the sample typically comprises a biological fluid sample of the organism (e.g. a human).
  • the biological fluid sample may be cerebrospinal fluid (CSF), urine, lymph, saliva, mucus or amniotic fluid.
  • CSF cerebrospinal fluid
  • the biological organism may be a commercially farmed animal, such as a fish, a horse, cattle, sheep or a pig; a pet, such as a cat or a dog; or a lab animal such as a mouse, a rat, a hamster or a guinea pig.
  • the plant may be a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa or cotton.
  • the plant may be a tree, such as Hymenoscyphus fraxineus.
  • the analyte may be an amino acid, a polypeptide, a carbohydrate (e.g. a polysaccharide or a disaccharide or a monosaccharide), lipids (fat), vitamin, a mineral, a metabolite or a polynucleotide.
  • the polypeptide may be a protein.
  • the protein can be an enzyme, antibody, complement protein (immune reaction), hormone, growth factor or growth regulatory protein, such as a cytokine, a structural protein (such as actin), a cellular receptor proteins (MHC), a transporter protein, glycosylated proteins.
  • the protein may be a bacterial protein, a fungal protein, a viral protein, a plant protein, an animal protein, a protista protein or a parasite-derived protein.
  • the analyte may be a biomarker.
  • the biomarker may be any biomarker known in the art that is capable of being bound by a probe.
  • the analyte may be interleukin-6, which is a marker of sepsis; procalcitoninin, which is a marker of a bacterial infection etc.
  • the sample may be a non-biological sample or a chemical sample.
  • the non-biological sample is preferably a fluid sample, e.g. a liquid sample or gaseous sample.
  • a non- biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.
  • the invention may be carried out on a sample that is known to contain or suspected to contain the analyte.
  • the invention may be carried out on a sample that contains one or more analytes whose identity is unknown.
  • the invention may be carried out on a sample to confirm the identity and/or concentration of one or more analytes whose presence in the sample is known or expected.
  • a method of tagging a polynucleotide with an azide group comprising: contacting a polynucleotide comprising a TCGA target site with an azide donor in order to tag the target site of the polynucleotide with an azide group.
  • the polynucleotide may be a double-stranded polynucleotide referred to herein, or a singlestranded polynucleotide.
  • An azide donor is a substance comprising an azide group with the ability to freely donate the azide group without reacting to other groups on the recipient molecule.
  • the azide donor may be RAdoHcy-8-Hy-PEG-N3, a modified SAM molecule containing an azide group or an azide modified nucleotide.
  • the contacting step may be performed in the presence of a catalyst, such as an enzyme.
  • the enzyme may be a methyl transferase, preferably Taql methyl transferase (EC 2.1.1.72)
  • the contacting step may be performed in the presence of a methyl transferase for at least about 20 minutes, at least about 30 minutes, at least about 30 minutes.
  • the contacting step may be performed in the presence of a methyl transferase (e.g.Taql methyl transferase) for between about 20 minutes and 24 hours.
  • a methyl transferase e.g.Taql methyl transferase
  • the contacting step is performed in the presence of a methyl transferase for between about 30 minutes and 2 hours, or between about 30 minutes and 90 minutes.
  • the contacting step may be performed in the presence of a methyl transferase (e.g. Taql methyl transferase) at about 34°C to about 55°C, preferably at about 45°C to about 55°C. Most preferably the contacting step is performed in the presence of a methyl transferase for about 30 minutes to about 90 minutes at about 50°C.
  • a methyl transferase e.g. Taql methyl transferase
  • one or more can mean two or more, three or more, four or more, five or more, six more, seven or more, eight or more, nine or more, 10 or more, 15 or more, or 20 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more or 1000.
  • the term “one or more” can alternatively mean “all”.
  • a conjugation site or a probe conjugation site may comprise “a nucleotide sequence specific for a transferase enzyme”.
  • a nucleotide sequence specific for a transferase enzyme can refer to a nucleotide sequence that comprises nucleotides that can be (specifically) modified by a transferase enzyme, e.g. a CpG island or a CpG thereof.
  • CpG island may refer to a region of DNA that comprises a large number of CpG dinucleotide repeats.
  • the region of DNA may be at least 200 nucleotides in length and have a CpG% of at least 50%.
  • a “(functional) fragment” thereof can refer to an analyte-binding fragment.
  • the meaning of the term “probe” as used herein depends on the context it is used in.
  • the probe in the context of a system for diagnosing an oral disease in a subject, the probe is a device configured to collect a fluid sample from an oral cavity of the subject.
  • the probe in the context of a method of making an immature conjugate subunit, the probe may be any agent that selectively or specifically binds to an analyte.
  • Figure 1 is a schematic drawing showing the process of detecting target analytes in a fluid sample obtain from the oral cavity of a subject, in accordance with an embodiment of the invention.
  • KD 10' 10 M (typical value for antibody/antigen interactions)
  • c(carrier) 10' 9 M
  • Left axis bar chart, concentration values in saliva.
  • Right axis fraction of capture probe bound to target, for determining the target concentration.
  • FIG 3 is a schematic overview of a method of making a modular double-stranded polynucleotide according to the invention (the method is also referred to herein as Sterically Controlled Nuclease Enhanced DNA Assembly (SCoNE DNA Assembly, Formally IADL));
  • Figure 4 is a 1 % agarose gel run at 75V for 45 minutes showing an N of 1 SCoNE.
  • Gene Ruler 1kbp (2) Gene Ruler low range, (3) 200bp DNA fragment (NoLimit, Thermo ScientificTM), (4) 2kbp DNA fragment (NoLimit, Thermo ScientificTM), (5) Negative control, (6) IADL 1pM, (7) Positive assembly fragment, and (8) IADL 5pM;
  • Figure 5 is a 1% agarose gel run at 75V for 45 minutes showing an N of 2 SCoNE experiment.
  • Gene Ruler 1 kb (2) Gene Ruler low range, (3) 2kbp DNA fragment (NoLimit, Thermo ScientificTM), (4) Short P1 strand (self-assembly check), (5) IADL (SCoNE) 1 pM, (6) Positive assembly fragment, (7) Negative control, and (8) IADL (SCoNE) 5pM;
  • Figure 6 is an agarose gel run at 75V for 60 minutes showing an N of 3 and 4 SCoNE experiment.
  • Figure 7 shows a Gibson fragment assembly highlighting the probe attached fragments (p) and the spacer fragments (s) of dsDNA. It also illustrates that the strand will not circularise due to the break formation between probe fragment 1 and spacer fragment 10;
  • Figure 8 is (A) an illustration of the DNA double helix backbone with adjoining capture probes for metabolites (probes A1 and A3 are aptamers; probes A2 and A5 are singlestranded DNA or RNA; and probe A4 is an antibody that binds a protein).
  • probes A1 and A3 are aptamers; probes A2 and A5 are singlestranded DNA or RNA; and probe A4 is an antibody that binds a protein).
  • B The experimental setup highlighting the differences in the signal obtained using a carrier-enhanced resistive pulse sensing technology when: (B1) no probe is bound, (B2) the analyte is not bound to the probe, and (B3) the analyte is bound to the probe.
  • Figure 9 is a 1 % agarose gel highlighting the ability to create decamer SCoNE structures (lanes 6 and 7).
  • Lane 1 Gene Ruler 1 kbp Thermo ScientificTM
  • lane 2 2kbp fragment NoLimit, Thermo ScientificTM
  • lane 3 10kbp fragment NoLimit, Thermo ScientificTM
  • lane 4 whole A DNA Thermo ScientificTM
  • lane 8 negative control we show our ability to generate decamer structures (lanes 6 and 7, 18.1).
  • Figure 10 shows a 1 % agarose gel run at 75V for 45 minutes which shows that the inventors were are able to extract biotin labelled p strands (yellow box, left hand box) from the reaction mixture (red box, right hand box) with lane 1 , gene ruler, lane 4 biotin extracted p strands, lane 6 reaction mixture prior to assembly.
  • Figure 11 shows a 1% agarose gel run at 75V for 45 minutes illustrating the inventors ability to assemble, and extract 3mer scone structures (yellow box, left hand box) from an assembled sample (red box, middle box). This also shows the need for an extraction method comparing to the starting reaction mixture (blue box, right hand box).
  • Figure 12 show respective intensity compared to biotin group positioning.
  • Figure 13 shows ELISA and newly adapted ELISA protocols.
  • Panel A shows a normal ELISA using the 3mer SCoNE structure for protein isolation using streptavidin to bind reactive biotin groups.
  • A1 shows SCoNE bound protein binding to primary IL6 antibody.
  • A2 shows Secondary IL6 antibody binding to protein.
  • A3 shows ABC binding to biotin labelled antibody.
  • A4 shows ABC converting TMB buffer to coloured compound for measurement
  • Panel B shows an ELISA using the 3mer SCoNE structure, but removing the secondary antibody and measuring the protein concentration based on the biotin binding alone.
  • B1 shows SCoNE bound protein binding to primary IL6 antibody.
  • B2 shows Secondary IL6 antibody not added.
  • B3 shows ABC binding to biotin labelled SCoNE.
  • B4 shows ABC converting TMB buffer to coloured compound for measurement.
  • Panel C shows a modified ELISA using the 4mer SCoNE structure where the primary antibody binds procalcitonin and the secondary binds IL6.
  • C1 shows SCoNE bound protein binding to primary procalcitonin antibody.
  • C2 shows Secondary IL6 antibody binding to IL6 protein
  • C3 shows ABC binding to biotin labelled IL6 antibody.
  • C4 shows ABC converting TMB buffer to coloured compound for measurement.
  • Panel D shows a similar experiment to C using the 4mer SCoNE structure where the structure has not been incubated with IL6, therefore the secondary antibody cannot bind and no signal should be observable.
  • D1 shows SCoNE bound protein binding to primary procalcitonin antibody.
  • D2 shows Secondary IL6 antibody added but cannot bind anything so is washed off
  • D3 shows ABC cannot bind as biotin sites are blocked by streptavidin.
  • D4 shows ABC not present so cannot convert TMB
  • Figure 14 shows percentage of SCoNE structures retained against expected during ELISA analysis.
  • Antibody secondary as referred to in the key of figure 14 corresponds to the left most bar of the graph.
  • “Streptavidin linker only” as referred to in the key of figure 14 corresponds to the second bar from the left of the graph.
  • “Separate protein linker IL6” as referred to in the key of figure 14 corresponds to the third bar from the left of the graph.
  • “Separate protein linker IL6 run 2” as referred to in the key of figure 14 corresponds to the fourth bar from the left of the graph.
  • “Separate protein linker Pro” as referred to in the key of figure 14 corresponds to the fifth bar from the left of the graph.
  • “Separate protein linker no IL6” as referred to in the key of figure 14 corresponds to the sixth bar from the left of the graph.
  • Figure 15 shows structure of the 4mer with biotin at positions 1 and 2, bound human IL6 on an aptamer at position 3 and a blank Procalcitonin at position 4.
  • Figure 16 shows all 4mer translocation events at different biases (V).
  • Figure 17 shows an inverse relationship between bias and event duration.
  • “-0.5” as referred to in the key of figure 17 corresponds to the left most data point on the graph, indicated by “x”.
  • “-0.6” as referred to in the key of figure 17 corresponds to the second data point from the left on the graph, indicated by “x”.
  • “-0.7” as referred to in the key of figure 17 corresponds to the third data point from the left on the graph, indicated by “x”.
  • “-0.8” as referred to in the key of figure 17 corresponds to the fourth data point from the left on the graph, indicated by “x”.
  • the values referred to in the key of figure 17 correspond to the bias voltage applied. Accordingly, reference to “-0.5”, as referred to in the key of figure 17, refers to “-0.5V”. As would be clear to the skillled person, reference to “-0.6” in the key of figure 17 thus refers to “-0.6V” and so on.
  • Figure 18 shows a comparison between SCoNE DNA average events at -0.6 and -0.7V (A and B) to bare DNA at the same biases (C and D).
  • Figure 19 shows a comparison of sub events analysis (frequency count) conducted between 4mer SCoNE DNA and bare 4kbp DNA fragments.
  • a comparison between sub event threshold (A) shows that increasing threshold value decreases the number of peaks observable (25- 175pA threshold respectively).
  • a 3D illustration of subevent position along the DNA backbone across a range of biases shows similar positioning of the subevents (B), most notably between -0.6 and -0.7V (C). The 2D representation re-enforced these conclusions (D).
  • Comparison between 4mer SCoNE DNA and bare 4kbp DNA fragments shows a lack of additional subevent peaks in the bare DNA samples represented in 3D and 2D views for direct analysis (E and F).
  • Figure 20 shows examples of SCoNE DNA current-time traces (A, E and I) for -0.5V, -0.6V, and -0.7V respectively, with single events highlighted for each bias (B-D, F-H, and J-L).
  • Figure 121 shows examples of bare 4kbp DNA current-time traces (A, E and I) for -0.5V, - 0.6V, and -0.7V respectively, with single events highlighted for each bias (B-D, F-H, and J-L).
  • Figure 1 shows a tip 10 of a probe which is used for obtaining a fluid sample from the oral cavity of a patient.
  • the tip 10 is tapered, and has a shape and size which is selected so that the tip 10 can be used to collect a fluid sample from a gingival crevice.
  • the tip houses a double-barrel nanopipette 12 (a theta pipette) comprising a first barrel 14 and a second barrel 16.
  • the first barrel 14 comprises a first electrode 18, and the second barrel 16 comprises a second electrode 20.
  • Each carrier molecule 22 comprises an elongate backbone 24, for example a DNA backbone. Immobilised on the backbone 24 are a plurality of capture moieties 26, which are specific for a target analyte (e.g. a protein biomarker) 28 that is indicative of gingivitis and/or periodontitis.
  • a target analyte e.g. a protein biomarker
  • the fluid sample is taken by applying a voltage to the first electrode 18, which causes fluid comprising the target analytes 28 to flow into the first barrel 14 by electroosmosis (step A).
  • the target analytes 28 are incubated with the carrier molecules 22 for a time sufficient to enable the capture moieties 26 to bind to the target analytes 28 (step B).
  • the application of a positive bias to the second barrel 16 relative to the first barrel 14 causes translocation of the carrier molecules 22 with capture target analytes 28 from the first barrel 14 to the second barrel 16, causing them to pass through nanopores 30a and 30b therebetween, thereby generating an electrical signal.
  • Figure 2 shows modelling results for target binding to a capture probe attached to a DNA carrier, for 12 target proteins. These proteins were identified in samples obtained from patients with different degrees of oral disease, ranging from healthy individuals to individuals with severe periodontal disease.
  • Left axis experimentally observed target concentrations across the patient population (mean +/- lower and upper bound).
  • Right axis probability of observing a translocating carrier with the target bound.
  • Simulation parameters carrier concentration: 10’ 9 M; dissociation constant for binding equilibrium: 10' 1 ° M.
  • the plot highlights the relevant concentration ranges (saliva) and provides an estimate of the probability of observing bound targets. It also illustrates how the approach can be employed to determine target concentrations, namely from the observed probability of "bound" events. The latter ranges from 0 - 100% and can be converted to concentration using the binding constant and the known binding equilibrium.
  • a nanopipette of 20-30 nm diameter may be prepared using the methods described by Loh et al., 2018, Anal Chem. 90, 14063-14071. Briefly, from filamented quartz capillaries (1 mm o.d., 0.5 mm i.d., 7.5 mm in length; Sutter Instruments). The capillaries contain a -160 pm glass filament that facilitates the filling of the nanopipette by capillary action. The glass capillaries are first plasma cleaned for 7 minutes (Harrick Scientific) before being loaded into a laser pipet puller (Sutter Instruments).
  • the inner diameter of the nanopipette can be estimated from the conductance of the pipette in 1 M KCI and/or using transmission electron microscopy (TEM) or optical microscopy.
  • TEM transmission electron microscopy
  • TEM imaging of the nanopipettes can be carried out using a JEOL JEM-21 OOF TEM.
  • the measurement of the images can be conducted using lmageJ.61.
  • Sample preparation may be carried out as follows: The tip of the pipet is positioned such that it is sitting parallel to the centre of the Cu TEM slot grid (catalogue no. GG030, Taab Laboratory Equipment Ltd.) and glued to the grid (e.g. using a two-component epoxy glue).
  • the glue is left to set (e.g. for 6 h), after which the pipette attached to the grid is cleaned under UV and ozone for 20 min (UVOCS). It is then sputter coated (Polaron Quorum Technologies) with 10 nm Cr to reduce charging effects.
  • the parts of the pipette lying just outside the grid may be cut off using a scalpel before the grid is placed in the sample holder of the TEM.
  • Double-barrel nanopipettes can be fabricated using the methods described in Saha-Shah et al,. Analyst (2016), 141 , 1958-1965 and Saha-Shah et al,. Chem. Sci., 2015,6, 3334-3341. Briefly, theta capillaries are pulled using a laser-based pipette puller (P-2000, Sutter Instrument, Novato, CA) and then a focused ion beam (Zeiss Auriga® Modular Cross Beam workstation Oberkochen, Germany, FIB), followed by milling each barrel to ⁇ 1 pm (internal diameter).
  • P-2000 laser-based pipette puller
  • a focused ion beam Zeiss Auriga® Modular Cross Beam workstation Oberkochen, Germany, FIB
  • Example 2 overview of a method of making a modular polynucleotide
  • FIG. 3 is a schematic overview of a method of making mature conjugate subunits, which in turn, can be used to create a modular polynucleotide (F) according to the invention.
  • a single aminated probe is conjugated to DBCO to create a modified probe for further conjugation.
  • B) Short, blunt-ended DNA fragments (less than 100 bp in length) are azidated using a methyl transferase (m.Taql), and an azide modified SAM.
  • C Independently, the modified probes and their respective azidated short, blunt-ended DNA fragments are combined together in a copper-free azide-alkyne reaction (a Click- iTTM reaction) to form of immature conjugate subunits.
  • a Click- iTTM reaction a copper-free azide-alkyne reaction
  • the immature conjugate subunits are then combined into a single vial along with spacer DNA strands (at least 100 bp in length), T5 exonuclease, Taq DNA Ligase, and Taq DNA polymerase.
  • E In this step, the 5’ ends of the spacer DNA strands are digested by T5 exonuclease to create sticky ends.
  • the 5’ ends of the immature conjugate subunits are digested by T5 exonuclease to create mature conjugate subunits (i.e. short, probe bound DNA fragments with sticky ends, which, in this case, are complementary to the sticky ends of the spacer DNA).
  • the T5 exonuclease is unable to navigate across the site at which the probe is attached to the polynucleotide of the immature conjugate and therefore gets knocked off.
  • the spacer DNA strands with sticky ends and the mature conjugate subunits are thus able to form bonds via their matching sticky ends.
  • DNA polymerase fills in any gaps created during digestion and DNA ligase seals the scars, forming phosphodiester bonds, thus resulting in the formation of a modular polynucleotide (a SCoNE structure) shown in (F).
  • DBCO-NHS-ester 1 mg of DBCO-NHS-ester added to 1ml of DMSO (HPLC grade) to create a 2.5mM stock b) Take 1 pl of the 2.5mM added to 49 pl of nuclease free water to create a working stock of 50pM
  • Probe strands a) Each probe strand should be constructed in a separate PCR tube, the volumes provided can be amplified as necessary. b) Pipette 4pl of azidated probe strand into a PCR tube c) Pipette 2 l of modified probe into the same tube d) Incubate at room temperature for a minimum 1.5 hours, can be left overnight. e) Can be stored at 4°C until required
  • Figure 4 is an agarose gel showing an N of 1 SCoNE (Sterically Controlled Nuclease Enhanced DNA Assembly), i.e. the method used to of make a modular double-stranded polynucleotide according to the invention.
  • the gel demonstrates that in lanes 6 and 8 there are bands above the 2kbp cut off (as indicated by the line using the gene ruler, lane 1, and the 2kbp fragment, lane 4) illustrating that it is possible to form a dimer (2 probe strands, 2 spacer strands) using the SCoNE technique. This is further shown by the absence of this band in lane 5 (negative control) highlighting that it is the conjugation that allows the formation of the larger structures.
  • the lower band at 1 kbp is unreacted spacer DNA.
  • Figure 5 is an agarose gel showing an N of 2 SCoNE experiments.
  • the gel demonstrates it is possible to generate a dimer (2 probe strands, 2 spacer strands) using the scone technique, as highlighted in lanes 5 and 8.
  • the absence of a fragment above 2kbp in lane 7 highlights that it is the use of the conjugates which allows for these fragments to be formed.
  • the 2kbp cut-off height is indicated by the line using the gene ruler, lane 1 , and the 2kbp fragment, lane 3.
  • the lower band at 1 kbp is unreacted spacer DNA.
  • Figure 6 is an agarose gel showing an N of 3 and 4 SCoNE experiments.
  • Lanes 2 and 3 (N3 and N4 respectively) highlight the formation of the dimer (2 probe strands, 2 spacer strands).
  • the absence of these fragments in the negative controls in lanes 4 and 5 (N3 and N4 respectively) highlight that it is the conjugation of the probes which allow for these fragments to be formed.
  • the 2kbp cutoff height is indicated by the line using the gene ruler, lane 1 , and the 2kbp fragment, lane 8.
  • the lower band at 1 kbp is unreacted spacer DNA.
  • Figure 9 is an agarose gel showing several intermediate steps are generated during the reaction (lanes 6 and 7, 18.2 and 18.3), which indicates the depletion of the starting DNA.
  • the absence of 1kbp DNA indicates that the spacer DNA has been fully incorporated into SCoNE structures unlike in the negative control (lane 8, B.5) where the 1kbp fragments are still present.
  • This example relates to isolation.
  • the nomenclature provided in the table immediately below is relevant.
  • experiments were performed to increase yield of single product collection, removing unwanted DNA fragments from the initial one pot reaction mixture, and to determine the most effective positioning of the biotin groups to allow for this.
  • the inventors have included an additional probe structure (DBCO-dPEG®12-biotin, Sigma- Aldrich) to assist with isolation.
  • the biotin groups added to the SCoNE structure were tested for their position effectiveness and the effectiveness of their use as a purification method.
  • the inventors were able to show that they can extract assembled SCoNE structures using the biotin tag they added in from the mix of starting elements. As is shown in figure 12, the inventors also demonstrated that the positioning of the biotin group is also important for extraction.
  • Scone structures are generated at different concentrations dependant on the vial used, therefore data is normalised to expected SCoNE concentration.
  • Nanodrop is used to determine starting SCoNE concentration.
  • 3mer and 4mer structures were generated with efficiencies of 57-61 %. Some of the SCoNE structures are also lost during extraction, this loss is approximately 20%. As the inventors extract twice, this is taken into account during calculations.
  • streptavidin was used and incubated for 30 minutes prior to experiments taking place. SCoNE structures were incubated for 30 minutes with the respective protein/s at 0.5ng/ml.
  • This example relates to translocation.
  • Translocation of bound SCoNE DNA through a nanopore was performed to assess firstly, the ability of the sensing apparatus to accurately detect DNA translocation. Secondly, to categorise the profile of SCoNE DNA and its differences from that of bare DNA. Finally, inclusion of a bound probe was utilised to determine the stability of the probe binding, translocation potential under real experimental conditions, and develop data analysis tools for subsequent comparison. The inventors successfully detected SCoNE DNA with bound analyte and successfully determined its differences to that of bare DNA.
  • Cleaned amber liquid cells were filled with 2ml of the 4 M LiCI 10% TE solution.
  • SCoNE DNA was added into the vial to achieve a final concentration of approximately 80pM.
  • a size- determined nanopipette was inserted into the cell, submerging the tip in the liquid.
  • Anodized silver/ silver chloride electrodes, soldered to gold contact pins, were added to the setup, such that one electrode sat inside the pipette chamber, and the other in the bulk solution, outside of the pipette. This was then attached to a custom low noise amplifier, sampling at 1 MHz.
  • a 100kHz in-line filter was attached to the output of the amplifier, and connected directly to a Picoscope 4262 oscilloscope, which was used for real-time monitoring of the system.
  • a custom MATLAB script was used to control the bias voltage applied to the system, which allowed for changing the input voltage during measurements. Each scan was saved for further event and sub event analysis performed by custom MATLAB scripts.
  • Sub event analysis provides a further insight into the substructure of the events as highlighted in figure 19.
  • the inventors applied a threshold for determining sub event analysis of between 25-175pA (10. A).
  • the DNA structure is approximately 4.1 kbp long, and comparisons are made between SCoNE 4mer and 4kbp DNA fragments (NoLimits).
  • DNA has the capability to translocate both forwards and backwards through the nanopore.
  • the size of the backbone has been normalised to values 0-1 with the relative positions of sub structures falling between these values. In a forwards translocation, the inventors expected to see peaks at positions near 0, 0.25, 0.5 and 0.75.
  • the inventors In a backwards translocation, the inventors expected to see peaks at positions 0.25, 0.5, 0.75 and near 1. As can be seen from the from figure 19A, there are several peaks which emerge from the events generated. In lower thresholds (25-75pA) the inventors observed peaks at near 0, near 0.25, 0.5, near 0.75 and some emergence near 1. Due to the size of the biotin binding groups, and the unbound aptamer, it is possible that during data acquisition some of the resolution near the beginning and end of the event is lost. When the inventors applied a threshold between 100- 150pA, the inventors observed loss of these initial, and ending peaks, however obtained a greater resolution of subevents at positions 0.25 and 0.5.
  • Figure 20 highlights a few events (excluding A, E and I) observed in the scans (A, E and I) across three biases used during experiments (- 0.5V, -0.6V and -0.7V respectively). These show a clear definition of sub events appearing from the baseline as opposed to noise contribution. This can be determined by comparison to bare 4kbp DNA translocation events (figure 21 , excluding A,E and I). Experiments were conducted using nanopores of similar size (usually between 10-20 nm pores, depending on the size of the analyte), with the only difference being the DNA in solution. It can also be noted that translocation frequency is much higher in bare 4kbp, this is due to a higher starting concentration.
  • the invention further includes the subject matter of the following numbered paragraphs (paras).
  • a method of making an immature conjugate subunit comprising: conjugating a (first) probe, for binding a (first) analyte, to a probe conjugation site of a
  • a method of making a mature conjugate subunit comprising: conjugating a (first) probe, for binding a (first) analyte, to a probe conjugation site of a (first) double-stranded polynucleotide, to create a (first) immature conjugate subunit; and forming a (first) mature conjugate subunit by cleaving the double- stranded polynucleotide of the (first) immature conjugate subunit to form a first sticky end, or a first sticky end and/or a second sticky end.
  • conjugating comprises contacting a polynucleotide having a probe conjugation site functionalised with an azide group with a probe functionalised with a DBCO- NHS ester.
  • the mature conjugate subunit is formed by cleaving the double-stranded polynucleotide of the immature conjugate subunit with an enzyme, optionally wherein the enzyme is a 5’ exonuclease, such as a T5 exonuclease.
  • annealing comprises contacting a (first) mature conjugate subunit with the (first) double- stranded polynucleotide spacer in the presence of a DNA ligase and a DNA polymerase.
  • a method of making a modular polynucleotide comprising: i. creating a mature conjugate subunit according to the method of any one of paragraphs 2 to 9;
  • annealing comprises contacting the two or more mature conjugate subunits in the presence of a DNA ligase and a DNA polymerase.
  • An immature conjugate subunit comprising: a probe, for binding an analyte, conjugated to a probe conjugation site of a double-stranded polynucleotide, wherein the probe conjugation site comprises a nucleotide sequence specific for a transferase enzyme.
  • a double-stranded modular polynucleotide comprising: a double-stranded polynucleotide backbone comprising a plurality of probe conjugation sites, each site having a nucleotide sequence specific for a transferase enzyme, wherein each probe conjugation sites is separated by at least 16 base pairs, and wherein at least one probe conjugation site is conjugated to a single probe for binding an analyte.
  • kits for determining if one or more analyte(s) is/are present in a sample comprising: i. a double-stranded polynucleotide spacer; and ii. an immature conjugate subunit according to any one of paragraphs 14 to 16 or an immature conjugate subunit made by the method of paragraph 1 ; or iii. a modular polynucleotide according to any one of paragraphs 17 to 20.
  • kits for diagnosing a test subject suffering from a medical condition comprising: i. a double-stranded polynucleotide spacer; and ii. an immature conjugate subunit according to any one of paragraphs 14 to 16 or an immature conjugate subunit made by the method of paragraph 1 ; or iii. a modular polynucleotide according to any one of paragraphs 17 to 20, wherein the presence of one or more analyte(s) in a bodily sample from a test subject is indicative that the subject suffers from the medical condition, or wherein the absence of the one or more analyte(s) from a bodily sample from a test subject is indicative that the subject suffers from the medical condition.
  • a method of determining if one or more analyte(s) is/are present in a sample comprising: i. contacting a modular polynucleotide according to any one of paragraphs 17 to 20 with a test sample; and then ii. analysing the modular polynucleotide using a carrier enhanced-resistance pulse sensing technology to determine if one or more analyte(s) is/are present in the test sample.

Abstract

La présente invention concerne un système et un dispositif destinés à être utilisés dans le diagnostic d'une maladie buccale. L'invention concerne également des procédés, des kits et des compositions destinés à être utilisés dans le diagnostic d'une maladie buccale. Plus particulièrement, l'invention concerne un système comprenant une sonde qui est conçue pour collecter un échantillon de fluide à partir de la cavité buccale d'un sujet, ainsi qu'un détecteur qui est conçu pour détecter dans l'échantillon de fluide la présence et/ou la concentration d'un analyte qui indique la maladie buccale.
PCT/GB2021/052870 2020-11-04 2021-11-04 Dispositif WO2022096893A1 (fr)

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US18/035,124 US20230408508A1 (en) 2020-11-04 2021-11-04 System and method for diagnosis of oral disease
EP21814839.3A EP4241084A1 (fr) 2020-11-04 2021-11-04 Dispositif
EP22712620.8A EP4314820A1 (fr) 2021-03-25 2022-03-24 Capture d'analyte
PCT/GB2022/050735 WO2022200793A1 (fr) 2021-03-25 2022-03-24 Capture d'analyte

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