SYSTEM AND METHOD FOR DIAGNOSIS OF ORAL DISEASE

Abstract

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.

Claims

1. A system for diagnosing an oral disease in a subject, wherein the system comprises: a probe which is configured to collect a fluid sample from an oral cavity of the subject; and a detector which is configured to detect in the fluid sample at least one of a presence and a concentration of an analyte which is indicative of the oral disease, wherein the detector is configured to detect at least one of the presence and the concentration of the analyte using resistive pulse sensing.

2. The system of claim 1, wherein the detector is in fluid communication with the probe.

3. The system of claim 1, wherein the detector and the probe are integrated into a single device.

4. The system of claim 1, wherein the probe comprises a tip which is configured to collect the fluid sample via at least one of suction, capillary action, and electroosmosis.

5. The system of claim 4, wherein the tip is configured for collecting gingival crevicular fluid (GCF) from a gingival crevice.

6. The system of claim 1, wherein the probe is a periodontal probe comprising a hollow tip.

7. The system of claim 1, wherein the detector comprises a nanopipette.

8. The system of claim 7, wherein the nanopipette is housed within the tip of the probe.

9. (canceled)

10. A composition comprising a plurality of carrier molecules for detecting of a target analyte using resistive pulse sensing, wherein each of the carrier molecules is functionalized with a capture moiety, wherein the capture moiety is capable of specifically binding to a biomarker of an oral disease.

11. The composition of claim 10, wherein the carrier molecules are nucleic acids (c.g. DNA).

12. The composition of claim 10, wherein the capture moiety is an antibody.

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. A method of diagnosing an oral disease in a subject, the method comprising using resistive pulse sensing to detect at least one of athe presence and a concentration of a target analyte in a fluid sample obtained from an oral cavity of a subject.

18. The method of claim 17, further comprising: a) applying a voltage across a nanopore to effect a translocation of any molecules present in the fluid sample through the nanopore; b) detecting a signal generated by the translocation of the molecules through the nanopore; and c) optionally, comparing the signal detected with a reference signal.

19. The method of claim 18, further comprising incubating the fluid sample with a plurality of carrier molecules prior to translocation, wherein the carrier molecules are functionalized with a capture moiety which is capable of specifically binding to the target analyte.

20. The method of claim 17, wherein the method is carried out using a probe configured for collecting the fluid sample from the oral cavity of the subject, wherein the probe comprises a hollow tip in which is housed a nanopipette.

21. The method of claim 20, wherein the probe is a periodontal probe.

22. The method of claims 17, wherein the fluid sample is selected from the group consisting of saliva andef gingival crevicular fluid (GCF).

23. The system method of claim 17, wherein the oral disease is periodontal disease.

24. The system of claim 1, wherein the detector is coupled to the probe.

25. The system of claim 1, wherein the oral disease is periodontal disease.

Description

[0274] Embodiments of the invention will now be described by way of example and with reference to the accompanying figures, in which:

[0275] FIG. 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; and

[0276] FIG. 2 shows simulation results based equilibrium binding theory with one target analyte binding site per carrier molecule (equilibrium constant KD=10.sup.10 M (typical value for antibody/antigen interactions), c(carrier)=10.sup.9 M), for upper, mean and lower concentration values of 12 identified marker proteins. Left axis: bar chart, concentration values in saliva. Right axis: fraction of capture probe bound to target, for determining the target concentration.

[0277] 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));

[0278] FIG. 4 is a 1% agarose gel run at 75V for 45 minutes showing an N of 1 SCoNE. (1) Gene Ruler 1 kbp, (2) Gene Ruler low range, (3) 200 bp DNA fragment (NoLimit, Thermo Scientific), (4) 2 kbp DNA fragment (NoLimit, Thermo Scientific), (5) Negative control, (6) IADL 1 pM, (7) Positive assembly fragment, and (8) IADL 5 pM;

[0279] FIG. 5 is a 1% agarose gel run at 75V for 45 minutes showing an N of 2 SCoNE experiment. (1) Gene Ruler 1 kb, (2) Gene Ruler low range, (3) 2 kbp DNA fragment (NoLimit, Thermo Scientific), (4) Short P1 strand (self-assembly check), (5) IADL (SCoNE) 1 pM, (6) Positive assembly fragment, (7) Negative control, and (8) IADL (SCoNE) 5 pM;

[0280] FIG. 6 is an agarose gel run at 75V for 60 minutes showing an N of 3 and 4 SCoNE experiment. (1) Gene Ruler 1 kbp, (2) IADL (SCoNE) 1pM, (3) IADL (SCoNE) 0.5 pM, (4) IADL (SCoNE) 0.25 pM, (5) IADL (SCoNE) 0.125 pM, (6) Positive Control, (7) Negative control, and (8) 2 kbp fragment (NoLimit, Thermo Scientific);

[0281] FIG. 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; and

[0282] FIG. 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 single-stranded 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.

[0283] FIG. 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 Scientific), lane 2 2 kbp fragment (NoLimit, Thermo Scientific), lane 3 10 kbp fragment (NoLimit, Thermo Scientific), lane 4 whole DNA (Thermo Scientific), lane 5 PCR amplified construct, lane 6 decamer SCoNE 1 pM (N=1), lane 7 decamer SCoNE 1 pM (N=2), lane 8 negative control. We show our ability to generate decamer structures (lanes 6 and 7, 18.1).

[0284] FIG. 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.

[0285] FIG. 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).

[0286] FIG. 12 show respective intensity compared to biotin group positioning.

[0287] FIG. 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 to colour.

[0288] FIG. 14 shows percentage of SCoNE structures retained against expected during ELISA analysis. Antibody secondary as referred to in the key of FIG. 14 corresponds to the left most bar of the graph. Streptavidin linker only as referred to in the key of FIG. 14 corresponds to the second bar from the left of the graph. Separate protein linker IL6 as referred to in the key of FIG. 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 FIG. 14 corresponds to the fourth bar from the left of the graph. Separate protein linker Pro as referred to in the key of FIG. 14 corresponds to the fifth bar from the left of the graph. Separate protein linker no IL6 as referred to in the key of FIG. 14 corresponds to the sixth bar from the left of the graph.

[0289] FIG. 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.

[0290] FIG. 16 shows all 4mer translocation events at different biases (V).

[0291] FIG. 17 shows an inverse relationship between bias and event duration. 0.5 as referred to in the key of FIG. 17 corresponds to the left most data point on the graph, indicated by x. 0.6 as referred to in the key of FIG. 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 FIG. 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 FIG. 17 corresponds to the fourth data point from the left on the graph, indicated by x. The values referred to in the key of FIG. 17 correspond to the bias voltage applied. Accordingly, reference to 0.5, as referred to in the key of FIG. 17, refers to 0.5V. As would be clear to the skillled person, reference to 0.6 in the key of FIG. 17 thus refers to 0.6V and so on.

[0292] FIG. 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).

[0293] FIG. 19 shows a comparison of sub events analysis (frequency count) conducted between 4mer SCoNE DNA and bare 4 kbp DNA fragments. A comparison between sub event threshold (A) shows that increasing threshold value decreases the number of peaks observable (25-175 pA 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 4 kbp 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).

[0294] FIG. 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).

[0295] FIG. 121 shows examples of bare 4 kbp 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).

[0296] FIG. 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.

[0297] Also located inside the first barrel 14 are carrier molecules 22. For simplicity, only a single carrier molecule is shown in FIG. 1, but it will be appreciated that a plurality of carrier molecules will be present. 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.

[0298] 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). Once inside the first barrel 14, 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).

[0299] Following incubation, 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.

[0300] FIG. 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.sup.9 M; dissociation constant for binding equilibrium: 10.sup.10 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.

EXAMPLE 1

Nanopipette Fabrication and Characterisation

Single-Barrel Pipettes

[0301] 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 m 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).

[0302] 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.

[0303] TEM imaging of the nanopipettes can be carried out using a JEOL JEM-2100F TEM. The measurement of the images can be conducted using ImageJ.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 Pipettes

[0304] Double-barrel nanopipettes (also known as theta pipettes) 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 m (internal diameter).

EXAMPLE 2OVERVIEW OF A METHOD OF MAKING A MODULAR POLYNUCLEOTIDE

[0305] 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. As shown in step (A), 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.Taq1), 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-iT reaction) to form of immature conjugate subunits. (D) 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. In addition, 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).

EXAMPLE 3PROTOCOL FOR CREATING A MODULAR POLYNUCLEOTIDE

Materials and Methods

Stock Concentrations

50 M DBCO-NHS-ester

[0306] a) 1 mg of DBCO-NHS-ester added to 1 ml of DMSO (HPLC grade) to create a 2.5 mM stock

[0307] b) Take 1 l of the 2.5 mM added to 49 l of nuclease free water to create a working stock of 50 M

1. Creating the Modified Probe

[0308] a) Each probe should be modified in a separate PCR tube, the volumes provided can be amplified as necessary

[0309] b) Pipette 18 l of 50 mM DBCO-NHS-ester into a PCR grade nuclease free vial

[0310] c) Add 2 l of prepared amine linked capture probe of interest (prepared as indicated by the manufacturer)

[0311] d) Incubate at room temperature for a minimum of 2 hours-overnight

[0312] e) Modified probes using ssDNA and aptamers are stable at 23 C. for 5 days.

2. Creating Modified Probe Strands (Azidation)

[0313] a) Each probe strand should be modified in a separate PCR tube, the volumes provided can be amplified as necessary

[0314] b) Set up the PCR tube with the following reactants and their respective volume, ensuring that the m.Taq1 enzyme is the final component to be added. Final volume is 20 l. This can be scaled up dependant on the requirements.

TABLE-US-00001 Reactant Volume (l) DNA strand (600 ng/ul) 1 Cutsmart buffer 2 (1 buffer comprises: 50 mM potassium acetate 20 mM Tris-acetate 10 mM magnesium acetate 100 g/ml BSA pH 7.9 at 25 C.) RAdoHcy-8-Hy-PEG-N3 (AW39) (or a 1 modified SAM molecule containing an azide group) m.Taq1 0.5 Nuclease free water 15.5

[0315] c) Incubate reaction vessel at 40 C. for 2 hours

[0316] d) Pipette 0.5 l of Protinase K (18 mg/ml) into the vial

[0317] e) Incubate at 50 C. for 1 hour

[0318] f) Allow to cool to room temperature for 20 minutes

[0319] g) Perform PCR clean-up protocol (GenElute Sigma-Aldrich) [0320] a. Ensure elution is into 20 l of either elution buffer or nuclease free water [0321] b. Concentration should be determined at this point (e.g. using Nanodrop)

[0322] h) Can be stored at 4 C. until required

3. Full Construction of Probe Strands

[0323] a) Each probe strand should be constructed in a separate PCR tube, the volumes provided can be amplified as necessary.

[0324] b) Pipette 4 l of azidated probe strand into a PCR tube

[0325] c) Pipette 2 l of modified probe into the same tube

[0326] d) Incubate at room temperature for a minimum 1.5 hours, can be left overnight.

[0327] e) Can be stored at 4 C. until required

4. Assembly

[0328] a) All components should be added into the same PCR vial for assembly.

[0329] b) Pipette equal concentrations of the reagents into a PCR tube to create the IADL-mastermix.

[0330] The current set up uses the following volumes due to concentration;

TABLE-US-00002 Volume to add (ul) Strand Volume to add (ul) Spacer1 1.5 Probe1 1 Spacer2 2 Probe2 1 Spacer3 1.5 Probe3 1 Spacer4 2 Probe4 1 Spacer5 1.5 Probe5 1 Spacer6 2 Probe6 1 Spacer7 1.5 Probe7 1 Spacer8 1.5 Probe8 1 Spacer9 1.5 Probe9 1 Spacer10 2 Probe10 1

[0331] c) Ensure the IADL-mastermix is mixed well using a pipette

[0332] d) In a new PCR vial pipette 10 l of the IADL-mastermix

[0333] e) Pipette 5 l of nuclease free water and mix well

[0334] f) Pipette 5 l of Gibson assembly mastermix [mastermix comprises a T5 exonuclease, a progressive polymerase (such as Taq polymerase) and Taq ligase (New England BioLabsGibson Assembly Master Mix/Gibson AssemblyCloning Kit-NEB # E2611S/L, # E5510S], and mix well (half of recommended)

[0335] g) Incubate at 40 oC for 1.5 hours (can be incubated at this temperature overnight)

[0336] h) Desired product has been isolated

5. Current Isolation Technique

[0337] a) Run the product on a 1% agarose gel at 80V for 80 minutes in 1 TAE buffer

[0338] a. The product can now be isolated by size comparison (at approximately 0.5 cm from pipetting well site)

[0339] b) Excise the DNA band from the gel using a sterile scalpel

[0340] c) Perform Gel extraction (GenElute Gel Extraction Kit Sigma-Aldrich) [0341] a. Ensure elution is into 20 l of either elution buffer or nuclease free water [0342] b. Final concentration can be determined now

[0343] d) Product is available for use

EXAMPLE 4CONFIRMATION OF MATURE CONJUGATE SYNTHESIS

[0344] FIG. 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 2 kbp cut off (as indicated by the line using the gene ruler, lane 1, and the 2 kbp 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.

[0345] FIG. 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 2 kbp in lane 7 highlights that it is the use of the conjugates which allows for these fragments to be formed. The 2 kbp cut-off height is indicated by the line using the gene ruler, lane 1, and the 2 kbp fragment, lane 3. The lower band at 1 kbp is unreacted spacer DNA.

[0346] FIG. 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 2 kbp cutoff height is indicated by the line using the gene ruler, lane 1, and the 2 kbp fragment, lane 8. The lower band at 1 kbp is unreacted spacer DNA.

[0347] FIG. 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 1 kbp DNA (lanes 6 and 7, B.4) indicates that the spacer DNA has been fully incorporated into SCoNE structures unlike in the negative control (lane 8, B.5) where the 1 kbp fragments are still present.

EXAMPLE 5

[0348] As described above, the inventors have shown that they are able to create structures with the expected weight. This was shown in 1% agarose gels.

[0349] This example relates to isolation. The nomenclature provided in the table immediately below is relevant. In this example, 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.

TABLE-US-00003 Nomenclature Explanation S Spacer DNA strand (1000 bp) P Probe DNA strand with aptamer attached (60 bp) B Probe DNA strand with biotin group attached (60 bp) 3mer A SCoNE structure comprised of 3S and 3P or B strands 4mer A SCoNE structure comprised of 4S and 4P or B strands 3S, 1P.sub.3, 2B.sub.1, 2 Indicates 3 S strands, 1 P strand at the third position, and 2 B strands at positions 1 and 2

[0350] The inventors have included an additional probe structure (DBCO-dPEG12-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 found that positions 1 and 2 of the final structure provided the highest yield.

Isolation

[0351] First, the inventors directly tested their ability to create the new biotin structures and tested their isolation method for these. Lane gaps between samples and the ruler were added to increase resolution of the low concentration samples as shown in FIG. 10.

[0352] From these results the inventors were able to show they can create p strands containing biotin and extract them from starting materials prior to the assembly step.

[0353] The positioning of the biotin group within the SCoNE structure was then compared with the achieved yield. Due to working in very low concentrations it was difficult to use gel electrophoresis for complete quantitation, however Gray values were compared across all groups tested as shown in FIG. 11.

[0354] From these results, 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 FIG. 12, the inventors also demonstrated that the positioning of the biotin group is also important for extraction.

[0355] This difference shows that biotin at positions 1 and 2 provide the highest efficiency in extraction. This could be due to multiple bindings to streptavidin during extraction assisting the binding of the SCoNE structure. The inventors observed a decrease in binding of 2.60%, 2.88%, 1.21%, and 2.48% respective to 3S,1P.sub.3,2B.sub.1,2.

Testing Binding Capacity

[0356] To perform binding capacities and efficiency of final structure formation, one standard (A) and three custom designed (B-D) ELISAs were performed, illustrated in FIG. 13. Several wash steps were performed between steps.

Notes on Extraction and Utilisation of SCoNE Structures

[0357] 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. Through calculating yields of SCoNE structures through the gel experiments, 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. Where applicable to bind the biotin groups on the SCoNE structure, an excess of 300%, to the expected concentration, 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 0.5 ng/ml. SCoNE structures from different assembly reactions were used for each experiment to ensure reliability in the assembly and isolation protocols. All experiments were completed to N=6. Expected concentrations were 54.45, 54.49, 21.50, and 13.82 ng/ml (for I L6 run 2, procalcitonin, and no I L6 experiments).

Results and Discussion

[0358] From the results of the ELISA performed, the inventors showed that they were able to successfully isolate SCoNE structures with functional capture groups. Results from the ELISAs are split into the 3mer and 4mer experiments. With respect to the starting concentrations expected, the yield of producing and successfully binding the proteins of interest are 71.31% (ELISA A), 67.49% (ELISA B), 54.00% (ELISA C), 74.88% (ELISA C), and 65.91%(ELISA C, using the reverse antibody set; IL6 primary with procalcitonin secondary). When removing the I L6 protein for ELISA D, a yield of 1.80% was observed. This indicates that not all biotin sites were fully occupied, therefore an error of 1.8% can be applied to all values obtained, bar ELISA B experiments.

EXAMPLE 6

[0359] 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.

Methods

[0360] Cleaned amber liquid cells were filled with 2 ml of the 4 M LiCI 10% TE solution. SCoNE DNA was added into the vial to achieve a final concentration of approximately 80 M. 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 100 kHz 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.

Translocation Results

[0361] When translocating the 4mer structure, as indicated in FIG. 15, the inventors observed translocation of the 4mer structure as illustrated in FIG. 16.

[0362] The inventors also showed that translocation follows the expected trend (to bare DNA) with increasing bias directly correlated with a decrease in event duration, FIG. 17.

[0363] When comparing average translocation profiles, see FIG. 18, the inventors observed that whilst the SCoNE translocation events have significant current decreases at specific points along the translocation, the bare DNA has less structure to where sub events can occur. It was also observed that decreases in current are matched by similar returns to the positive in bare DNA, these positive spikes are not observed in SCoNE DNA. This is further highlighted when looking at singular events.

[0364] Sub event analysis provides a further insight into the substructure of the events as highlighted in FIG. 19. The inventors applied a threshold for determining sub event analysis of between (10.A). The DNA structure is approximately 4.1 kbp long, and comparisons are made between SCoNE 4mer and 4 kbp DNA fragments (NoLimits). DNA has the capability to translocate both forwards and backwards through the nanopore. In several of the figures herein, 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. 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 FIG. 19A, there are several peaks which emerge from the events generated. In lower thresholds (25-75 pA) the inventors observed peaks at near 0, near 0.25, 0.5, near 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 100-150 pA, the inventors observed loss of these initial, and ending peaks, however obtained a greater resolution of subevents at positions 0.25 and 0.5. Above 150 pA threshold the inventors observed only the peak at 0.25 remaining, indicating they had filtered out useful subevents. When the inventors use a 100 pA and visualise the event across the different biases applied (see FIG. 19B) they observed that there are clear peaks across the datasets at positions 0.5, and potentially near 1. This is even visible in the 0.5V bias which had a very limited number of translocations. These peaks are most notable in the 0.6 and 0.7V biases (see FIGS. 19C, and 19D). When the inventors compared the subevent analysis to bare 4 kbp DNA (nearest comparable size), they observed that the bare DNA creates a smooth shape across the event, lacking notable subevent peaks across the backbone (see FIGS. 19.E and 19.F). Due to differences in starting concentration, frequency counts have been normalised against total counts for direct comparison.

Single Event View

[0365] When the inventors viewed singular events, they observed the trends observed in both the average analysis and the subevent analysis. FIG. 20 highlights a few events (excluding A, E and I) observed in the scans (A, E and I) across three biases used during experiments (-0.6V 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 4 kbp DNA translocation events (FIG. 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 4 kbp, this is due to a higher starting concentration.

SUMMARY

[0366] Overall, the inventors have: [0367] demonstrated that SCoNE DNA can be translocated and sub-events can be detected successfully; [0368] demonstrated that SCoNE DNA-protein binding can be successfully achieved; [0369] developed a DNA extraction method and tested this successfully; [0370] developed new ELISA methods and tested these for protein binding with high efficiency; and [0371] compared SCoNE to bare 4 kbp DNA and shown a clear distinction between event shapes

EXAMPLE 7

[0372] The invention further includes the subject matter of the following numbered paragraphs (paras). [0373] 1. A method of making an immature conjugate subunit, the method comprising: [0374] 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. [0375] 2. A method of making a mature conjugate subunit, the method comprising: [0376] 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 [0377] 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. [0378] 3. The method according to paragraph 2, further comprising: [0379] 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. [0380] 4. The method according to any one of paragraphs 1 to 3, wherein conjugating comprises contacting a polynucleotide having a probe conjugation site functionalised with an azide group with a probe functionalised with a DBCO-NHS ester. [0381] 5. The method according to any one of paragraphs 1 to 4, wherein the polynucleotide of the immature conjugate subunit comprises a first blunt end and/or a second blunt end. [0382] 6. The method according to any one of paragraphs 2 to 5, wherein the polynucleotide of the double-stranded mature conjugate subunit is between about 20 and about 100 base pairs/nucleotides in length. [0383] 7. The method according to any one of paragraphs 2 to 6, wherein the double-stranded polynucleotide of the mature conjugate subunit comprises 5-sticky ends and/or 3-sticky ends. [0384] 8. The method according to any one of paragraphs 2 to 7, wherein 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. [0385] 9. The method according to any one of paragraphs 3 to 8, wherein 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. [0386] 10. A method of making a modular polynucleotide comprising: [0387] i. creating a mature conjugate subunit according to the method of any one of paragraphs 2 to 9; [0388] ii. repeating the method of any one of paragraphs 2 to 9 to create a total of two or more mature conjugate subunits, [0389] wherein each of the two or more mature conjugate subunits has a (first or second) sticky end that is complementary with a (first or second) sticky end of a separate mature conjugate subunit; and [0390] iii. annealing the complementary sticky ends of the two or more mature conjugate subunits to create a modular polynucleotide comprising a double-stranded polynucleotide backbone having two or more mature conjugate subunits. [0391] 11. The method according to paragraph 10, wherein annealing comprises contacting the two or more mature conjugate subunits in the presence of a DNA ligase and a DNA polymerase. [0392] 12. The method according to paragraph 9 or 11, wherein the DNA ligase is Taq ligase. [0393] 13. The method according to paragraph 9, 11 or 12, wherein the DNA polymerase is Taq DNA polymerase. [0394] 14. An immature conjugate subunit comprising: [0395] a probe, for binding an analyte, conjugated to a probe conjugation site of a double-stranded polynucleotide, [0396] wherein the probe conjugation site comprises a nucleotide sequence specific for a transferase enzyme. [0397] 15. The immature conjugate subunit according to paragraph 14, wherein the polynucleotide of the double-stranded mature conjugate subunit is between about 20 and about 100 base pairs/nucleotides in length. [0398] 16. The immature conjugate subunit according to paragraph 14 or paragraph 15, wherein the polynucleotide of the double-stranded mature conjugate subunit comprises a first blunt end or a second blunt end, and/or wherein the polynucleotide of the double-stranded mature conjugate subunit comprises 5-sticky ends or 3-sticky ends. [0399] 17. 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. [0400] 18. The immature conjugate according to any one of paragraphs 14 to 16 or the double-stranded modular polynucleotide according to paragraph 17, wherein the transferase enzyme is a methyl transferase, or wherein the nucleotide sequence specific for a transferase enzyme comprises a CpG island or a TOGA nucleotide sequence. [0401] 19. The double-stranded modular polynucleotide according to paragraph 17 or paragraph 18, wherein each of the probe conjugation sites is separated by at least about 30 base pairs. [0402] 20. The The method of any one of paragraphs 2 to 13, or the immature conjugate subunit according to any one of paragraphs 14 to 16, or the double-stranded modular polynucleotide according to any one of paragraphs 17 to 19, wherein the probe(s) is/are one or more selected from the group consisting of a single-stranded nucleotide, an antibody, a (functional) fragment of an antibody and/or an aptamer. [0403] 21. A kit for determining if one or more analyte(s) is/are present in a sample, the kit comprising: [0404] i. a double-stranded polynucleotide spacer; and [0405] 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 [0406] iii. a modular polynucleotide according to any one of paragraphs 17 to 20. [0407] 22. A kit for diagnosing a test subject suffering from a medical condition, the kit comprising: [0408] i. a double-stranded polynucleotide spacer; and [0409] 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 [0410] iii. a modular polynucleotide according to any one of paragraphs 17 to 20, [0411] 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. [0412] 23. The kit of paragraph 21 or 22, wherein the kit comprises a negative control and/or a positive control. [0413] 24. The kit of any one of paragraphs 21 to 23, wherein the kit comprises a buffer and/or one or more enzymes selected from the group consisting of a DNA polymerase, a nuclease (e.g. exonuclease) and a DNA ligase. [0414] 25. The A method of determining if one or more analyte(s) is/are present in a sample, the method comprising: [0415] i. contacting a modular polynucleotide according to any one of paragraphs 17 to 20 with a test sample; and then [0416] 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.