ANALYTE DETECTION METHOD

Abstract

The present invention provides a method of detecting one or more analytes in a target sample, the method comprising: a. providing a nanoparticle dimer adapted to bind the analyte; b. causing the dimer to pass through a nanopore by voltage-driven translocation; c. observing changes in the translocation current; and d. comparing the translocation current profile of the target sample to the translocation current profile of a control sample; wherein a change in the translocation current profile of the target sample versus the control sample indicates the presence of the analyte in the target sample. Also provided is a method of detecting one or more analytes in a target sample, the method comprising: a. providing a nanoparticle adapted to bind the analyte; b. providing a carrier nucleic acid molecule with at least one single-stranded region; c. contacting the carrier nucleic acid molecule and nanoparticle with the target sample, forming a carrier nucleic acid/analyte/nanoparticle complex; b. causing the carrier nucleic acid/analyte/nanoparticle complex to pass through a biological nanopore by voltage-driven translocation; c. observing changes in the translocation current; and d. comparing the translocation current profile of the target sample to the translocation current profile of a control sample; wherein a change in the translocation current profile of the target sample versus the control sample indicates the presence of the analyte in the target sample.

Claims

1. A method of detecting one or more analytes in a target sample, the method comprising: a. providing a nanoparticle dimer adapted to bind the analyte; b. causing the dimer to pass through a nanopore by voltage-driven translocation; c. observing changes in the translocation current; and d. comparing the translocation current profile of the target sample to the translocation current profile of a control sample; wherein a change in the translocation current profile of the target sample versus the control sample indicates the presence of the analyte in the target sample.

2. The method of claim 1, wherein the dimer comprises two nanoparticles linked by one or more nucleic acids, wherein at least one of the nucleic acids includes an aptamer specific for the analyte.

3. The method of claim 2, wherein the nanoparticles are linked by partially complementary nucleic acids.

4. The method of claim 1, wherein the dimer comprises two nanoparticles, each of which is attached to a nucleic acid, and each of the nucleic acids includes part of an aptamer specific for the analyte, such that in the presence of the analyte an aptamer is formed and thereby a dimer is formed.

5. The method of claim 1, wherein the analyte is a protein.

6. The method of claim 1, wherein the dimer comprises two nanoparticles, each of which is modified with a single stranded DNA (ssDNA), wherein one ssDNA includes a sequence which is complementary to the sequence of one end of the analyte, and the other ssDNA includes a sequence which is complementary to the sequence of the other end of the analyte, such that in the presence of the analyte a dimer is formed.

7. The method of claim 6, wherein the analyte is an miRNA.

8. The method of claim 1, wherein the dimer comprises two nanoparticles, each of which is conjugated to an antibody specific to a different epitope on the analyte, such that in the presence of the analyte a dimer is formed.

9. The method of claim 8, wherein the analyte is an antigen.

10. The method of claim 1, wherein the nanoparticles in the dimer are of substantially the same diameter.

11. The method of claim 1, wherein the nanoparticles in the dimer are of different diameters.

12. The method of claim 1, wherein the nanopore is the tip of a nanopipette.

13. A method of detecting one or more analytes in a target sample, the method comprising: a. providing a nanoparticle adapted to bind the analyte; b. providing a carrier nucleic acid molecule with at least one single-stranded region; c. contacting the carrier nucleic acid molecule and nanoparticle with the target sample, forming a carrier nucleic acid/analyte/nanoparticle complex; d. causing the carrier nucleic acid/analyte/nanoparticle complex to pass through a biological nanopore by voltage-driven translocation; e. observing changes in the translocation current; and f. comparing the translocation current profile of the target sample to the translocation current profile of a control sample; wherein a change in the translocation current profile of the target sample versus the control sample indicates the presence of the analyte in the target sample.

14. The method of claim 13, wherein the carrier nucleic acid molecule includes an aptamer specific for the analyte and the nanoparticle is conjugated to an antibody specific to a different epitope on the analyte from that to which the aptamer binds.

15. The method of claim 13, wherein the analyte is a protein.

16. The method of claim 13, wherein the carrier nucleic acid molecule is a ssDNA which includes a sequence that is complementary to the sequence of one end of the analyte and the nanoparticle is modified with a ssDNA which includes a sequence that is complementary to the sequence of the other end of the analyte.

17. The method of claim 16, wherein the analyte is an miRNA.

18. The method of claim 13, wherein the biological nanopore is alpha-hemolysin.

19. The method of claim 13, wherein the nanoparticle is a gold nanoparticle (AuNP).

20. The method of claim 13, wherein the target sample is a biological sample selected from blood, serum, lymph, sputum, urine, faeces, semen, sweat, tears, amniotic fluid, cerebrospinal fluid (CSF) and wound exudate.

Description

DETAILED DESCRIPTION

[0087] The ability to measure specific, selective biomarker molecules at single molecule level in physicians' surgeries and clinics has the potential to revolutionize disease diagnosis, monitoring, and therapy. Early and rapid diagnosis is an important factor to enhance the effectiveness of treatment. In many methods of early stage diagnosis, traditional biomarker detection methods like PCR and antigen-detecting ELISAs are widely used but limited due to many factors: 1) Low concentration of target biomarker molecules. People may be poor producers of an antibody or may have some interfering substance in their blood. The amount of antibody, consequently, may be too low to measure accurately or may go undetected. 2) Lack of single molecule data. This may cause the unspecific detection or recognise all isoforms of one same protein in a sample. 3) Other factors such as complex sample preparation and time consumption. Therefore, a fast, accurate and specific single molecule detection method is urgently needed.

[0088] Nanopores provide a label-free platform for sensing single biomolecules. Under applied potential, charged molecules will pass through the nanoscale pore and the resulting ionic current can be measured with standard electrophysiological techniques. Normally, long strand DNA molecules and large protein molecules can be distinguished easily by recognising the ionic current signal. However, this single molecule method is still challenging due to the low concentration in biological samples, fast translocation time of the analytes, poor analyte selectivity and low signal-to-noise ratio. Especially on the detection of protein molecules with small size and heterogeneous charge, such as lysozyme, thrombin and alpha synuclein, the single molecule signal becomes undetectable as the speed at which these molecules get transported through the nanopore is often too quick and hence hard to resolve. The present invention provides flexible, efficient and low-cost strategies to sense biomolecules of different sizes using a high resolution nanopore system.

[0089] The first aspect of the invention provides a series of molecular carriers and probes based on a nanoparticle dimer system, preferably a gold nanoparticle (AuNP) dimer system, which can deliver small analytes through a nanopore with improved signal-to-noise ratios.

[0090] The principle underlying the invention is increasing the effective size of the analyte using the nanoparticle dimer system. The signal of the dimer/analyte complex passing through a nanopore via voltage-driven translocation can be distinguished from the unbound dimer, allowing for detection even where the signal of the analyte cannot easily be detected.

[0091] The target analyte binds to dumbbell-shaped dimer carriers, with the corresponding aptamer located in the middle part, and then will be transported through a nanopore such as a fine-tuning nanopipette. Recorded by a high-bandwidth instrument, the high-res signal of nanoparticle monomers, nanoparticle dimers and nanoparticles with target protein can be differentiated by analysing the translocation events.

[0092] Moreover, this dimer system can be used to detect even smaller molecules which are highly likely undetectable in single molecule level. When the target protein is added into the solution, two nanoparticle monomers with different binding sites (such as antibody 1 and antibody 2) will link to each other and generate a nanoparticle-Antigen-nanoparticle dumbbell molecule because they will both bind to the target molecule. By detecting the ratio between the monomer and dimer, the high-res nanopore system not only can sense the antigens but also can quantify the trace amount concentration of them.

[0093] As described herein, the present invention is applicable to any nanoparticle which is suitable (in terms of its size and surface charge) for electrical detection when passed through a nanopore using voltage-driven translocation.

[0094] The sensing approaches of the present application are based on the differentiation of nanoparticles or nanoparticle-based conjugates. Herein, all nanoparticles with a suitable size and charge can be used in these methods. This includes nanoparticles formed of metals other than gold, alloys, polymers and silica. With different nanoparticles, the functionalised group on DNA may be changed to attach to it. Numerous nanoparticle (NP)/DNA conjugates, which can be used in the sensing system of the invention, have been reported (Samanta, A.; Medintz, I. L., Nanoparticles and DNA—a powerful and growing functional combination in bionanotechnology. Nanoscale 2016, 8 (17), 9037-9095) and examples of these are given in the table below.

TABLE-US-00002 Nanoparticle Constituents DNA AuNP Au FAM labeled T-rich DNA AuNR Au Leukemia T cell targeting SH-DNA Au/Ag hybrid Au/Ag Cytosine rich ssDNA AgNP Ag SH-DNA AgNC Few Ag atoms G-rich cocaine binding aptamer MNP Fe.sub.3O.sub.4 Thrombin binding SH-DNA aptamer PtNP Pt SH-DNA PdNP Pd Thiol and amine functionalized oligos QD CdSe/ZnS Dye-labeled photonic wire SWCNT Carbon Ce6 conjugated thrombin binding aptamer GO Carbon Short dsDNA with random sequence Micelle DNA + PPO ssDNA covalently attached to PPO Polyacrylamide-NP Polyacrylamide Dye-quencher labeled ATP aptamer Viral NP Bacteriophage Jurkat leukemia T cell MS2 capsid specific DNA aptamer Ferritin NPs hFTN-H/eGFP Amine modified PDGF or DsRed) specific aptamer UCNP Yb.sup.3+ or Tm.sup.3+ Amine-DNA with doped NaYF.sub.4 targeting sequence Chalcogenide-NP CuS Amine modified targeting DNA Alkaline earth Ca(H.sub.2PO.sub.4).sub.2, eGFP encoding metal NP CaHPO.sub.4 plasmid DNA Ca.sub.3(PO.sub.4).sub.2 DNANP DNA >100 short oligomers, Silane functionalised SiO2/Silane Amine modified DNA SiO2 NP

[0095] Abbreviations used in the table are as follows:

[0096] AuNP Gold nanoparticle

[0097] AuNR Gold nanorod

[0098] AgNP Silver nanoparticle

[0099] AgNC Silver nanocluster

[0100] MNP Magnetic nanoparticles

[0101] PtNP Platinum nanoparticle

[0102] PdNP Palladium nanoparticle

[0103] QD Quantum dot

[0104] SWCNT Single wall carbon nanotube

[0105] GO Graphene oxide

[0106] UCNP Upconversion nanoparticle

[0107] DNANP Deoxyribonucleic acid nanoparticle

[0108] In the present invention, the nanoparticle is typically a gold nanoparticle (AuNP). However, any of the other nanoparticles (NPs) described in the above table may alternatively be used.

[0109] There are many kinds of nanopore systems, including biological nanopore (e.g. alpha-hemolysin, MspA porin) and solid-state nanopore (e.g. Ion beam or electron beam drilled silicon nitrite membrane or graphene). In the Examples herein, nanopipettes were used among a variety of solid-state nanopore because of several advantages such as ease of fabrication, ease of set up (i.e., they can be tuned accurately) and low electrical noise. Nanopipettes may be manufactured by any suitable method available to the trained person. Quartz nanopipettes are particularly preferred as they are relatively easy to fabricate and do not introduce extra electrical noise or optical background.

[0110] Voltage-driven translocation through the nanopore may be achieved via any suitable means.

[0111] The ion currents of translocation are measured with the high-bandwidth amplifier such as a VC100 (Chimera Instruments). Typically, a grounded Faraday cage will be used to protect the nanopore system. For recording, a typical sampling rate is 1 MHz, and typically the data is filtered with 100 kHz low pass filter.

[0112] The inventors have designed a dumbbell-shaped nanoparticle dimer which is easy to fabricate and will cause specific signal shape when it passes through the nanopore. Ideally, a double peak event will be observed when a dimer molecule goes across the nanopore because the ionic signal will change while one nanoparticle is going into the pore and leaving. The dimer carrier will bind to the target protein molecule specifically by its aptamer branch. With the oligonucleotide sequences, aptamers are able to bind to their targets in a very specific way. Particularly, aptamers can be made to be applicable for almost any given target molecule, since aptamers can be collected through exponential enrichment process, in which ligands involved systematic evolution. Also, aptamers have other advantages such as low immunogenicity, small size, ease of modification and production and low toxicity. When a protein is transported by this dimer carrier, the changes of the double-peak event will give the information of this target molecule.

[0113] In one case, the nanoparticle monomer was based on AuNPs with 16-20 nm diameter with the surface functionalized by single stranded DNA (25-100 bases). However, nanoparticles of a variety of sizes may be used in the present invention. An AuNP dimer is 2 AuNP linked by a double stranded DNA. This dumbbell shape molecule can be self-assembled from two AuNP monomers with complementary ssDNA. The AuNP dimer protein carrier is a dimer with an aptamer branch in the middle. This dimer may be self-assembled from monomers with a ssDNA 1 (100 bases) and a ssDNA 2 (50 bases, DNA 2 is complementary with the end of DNA 1). The aptamer with DNA 3 (10-20 bases, DNA 3 is complementary with the unpaired part of DNA 1) then attached to the dimer. The resulting AuNPs dimer with a branch of aptamer can be the molecular carrier of a specific protein.

[0114] In an alternative embodiment, one nanoparticle (such as an AuNP) is functionalised with ssDNA with a part of aptamer sequence at the end while the other nanoparticle (such as an AuNP) is attached to ssDNA with the rest of the aptamer sequence; when the target protein is present, the two ssDNA parts of the aptamer will link together and form an aptamer which binds to the target protein, leading to the conjugation of the nanoparticle monomers. In this embodiment, the aptamer could be split in any proportion, as long as the parts link together to form a complete aptamer when the target protein is present.

[0115] It can therefore be seen that the aptamer may already be present in the DNA that is attached to one of the nanoparticles. Alternatively, the aptamer may be formed when the dimer forms, by hybridization of complementary single-stranded DNA that is bound to each of the nanoparticles.

[0116] By using the nanoparticle monomer probe, the system can be further extended to detect even smaller targets which are highly likely undetectable in single molecule level. Based on the result that the nanopore detection set up can distinguish nanoparticle monomers and dimers efficiently, the single molecule detection of small molecules can be achieved indirectly. Driven by the addition of target antigens, two nanoparticle monomers with different antibodies (both antibodies can bind to the antigen) will assemble to dimer by the strong interaction of antigen-antibody. To calculate the ratio of the double-peak signal caused by dimer molecule translocation to the single peak signal caused by monomer translocation, we can know the accurate concentration of the targeting molecule.

[0117] To detect an antigen, a pair of nanoparticle monomers (such as AuNP monomers) can be prepared in which one of the nanoparticle monomers (such as an AuNP monomer) links or is conjugated to antibody 1 while the other links or is conjugated to antibody 2 (Antibody 1 and 2 can bind to the antigen simultaneously). Once the target antigen added to the solutions containing probes with corresponding antibodies, dimer molecules will be generated by the antigen-antibody interaction. One or more of the antibodies may be modified, for example thiol functionalized.

[0118] Although antibodies are exemplified herein, it will be apparent that any antigen specific binding molecule that can be attached to a nanoparticle could be used in the present invention. In another embodiment, a pair of nanoparticle monomers (such as AuNP monomers) is prepared in which one of the nanoparticle monomers is functionalised with a ssDNA which includes a sequence which is complementary to the sequence of one end of the analyte, and the other nanoparticle monomers is functionalised with a ssDNA which includes a sequence which is complementary to the sequence of the other end of the analyte, such that in the presence of the analyte a dimer is formed. The two ssDNAs may include half of the complementary sequence each, or there may be an alternative arrangement in which, say the two ssDNAs include 40% and 60%, 30% and 70%, or any other percentage of the complementary sequence, as long as the nanoparticles dimerise on binding the target analyte. It can therefore be seen that the two nanoparticles dimerise via hybridisation of the ssDNAs attached to each of the nanoparticles to the target analyte, which is a nucleic acid. This embodiment is useful for detection of nucleic acid analytes, such as DNA or RNA, which may be single-stranded or double-stranded with a single-stranded overhang to allow for binding to the complementary sequence attached to the respective two nanoparticles. This embodiment is especially useful where the analyte is miRNA, but the analyte may be any kind of RNA such as mRNA, tRNA, siRNA, gRNA, ncRNA, exRNA, crRNA, or lncRNA.

[0119] While being the oldest type of nanopores, natural protein channels are still appealing because of several unique advantages such as high sensitivity and high resolution. However, biological nanopore biosensors are limited by their size, which is small and not tuneable. Thus, selective sensing of biomarkers in biological nanopore is still challenging.

[0120] The second aspect of the invention harnesses the potential of using nanopore based sensing techniques in biological nanopores. Inspired by the sensing of dimerization of nanoparticle probes, ssDNA and nanoparticle probes are designed. For single-molecule protein detection, a ssDNA with aptamer end and nanoparticle (such as AuNP) with antibody are used, see FIG. 13. With the presence of target protein, the ssDNA will link to the nanoparticle (such as AuNP) by an aptamer-protein-antibody bridge, FIG. 13a. In this case, the ssDNA act as a capture domain and the nanoparticle contributes to the signal amplification. The bacterial protein pore α-haemolysin (α-HL) is one of the most widely used biological channel in nanopore analytics. A ssDNA can translocate through α-HL nanopore (FIG. 13a) whereas protein cannot (FIG. 13b). However, the second level of protein blockade signal, which is highly dependent on the size and charge of the protein, may not be obvious. Herein, the nanoparticle (such as AuNP) can amplify this selective binding signal, see FIG. 13.

[0121] Based on the same principle, miRNA also can be detected by ssDNA and nanoparticle (such as AuNP) with specific sequence (FIG. 14). With the presence of the target miRNA, the ssDNA carrier with a complementary DNA (cDNA1) end to miRNA and the nanoparticle probe modified with the remainder of the complementary DNA (cDNA2) will self-assemble to a ssDNA-miRNA-nanoparticle complex due to DNA-RNA hybridisation. When free ssDNA translocates through the biological nanopore, the ionic current will be blocked by the DNA molecule and show a drop-down. Since the hybridised dsDNA cannot enter the nanopore, for ssDNA bound with target miRNA, the signal will show a subpeak due to the stuck or release of target miRNA. With the form of ssDNA-miRNA-nanoparticle complex, the subpeak will be amplified due to the existence of the nanoparticle (such as AuNP). The nanoparticle serves as a signal amplifier and effectively increases signal-to-noise ratio.

[0122] Embodiments described herein in relation to the first aspect of the invention are applicable to the second aspect of the invention mutatis mutandis.

[0123] The present invention will be further understood by reference to the following examples.

Examples

Materials & Methods

The Fabrication of Nanopipettes

[0124] The single barrel quartz capillaries (o.d., 1.0 mm, i.d., 0.7 mm, Intracell) were plasma cleaned (Harrick Plasma), and pulled with a laser-based P-2000 pipette puller (Sutter Instruments) using a two-line program (heat 800, filament 4, velocity 30, delay 170, and pull 80; heat 825, filament 3, velocity 20, delay 145, and pull 130) to produce nanopipettes with the nanopore diameters of approximately 34 nm at the tip as characterised by SEM imaging. It should be noted that the above pulling parameters are instrument specific and variations will exist from puller to puller.

The Fabrication of AuNP Based Nanostructures

[0125] The effective length of a nanopipette is the portion of the electrolyte-filled pore over which the majority (for our calculations 75-80%) of its ionic resistance is focused. In addition, the voltage drop is greatest in this area, resulting a strongest electric field. For a cylindrical nanopore, the pore length (On the solid state nanopore always been considered as the thick of the membrane) equals to the effective length of the nanopore. Theoretically, the voltage drops linearly along the pore length in these cylindrical pores. However, the conical nanopore, which located on the tip of the nanopipette, results a nonlinear drop of the electric field and resistance along with the pore length because the pore radius changes along the distance to the end.

[0126] To detect the nanopipette effective length, the resistance distribution along the pore axis should be analysed (Figure. 5). The resistance with different position (Rx) of the nanopore can be estimated. The distance from the end, x, is ranging from 0 to L, which is a length that long enough and can be studied from the SEM images. D0 represents the diameter of the end of the nanopipette part and the DL represents the diameter of the area which L from the end. The diameter along with the distance, Dx, can be calculated. Then the Rx can be estimated.

[0127] Dx can be expressed as the following function of x:

[00001]$\begin{array}{cc}{D}_x=\frac{x\left(D_L-D_0\right)+L{D}_0}{L}& \left(1\right)\end{array}$

In our calculation, the step of x is 1 nm.
The resistance Rx can be calculated as following function:

[00002]$\begin{array}{cc}{R}_x=\frac{4L\rho }{\pi D_0{D}_x}& \left(2\right)\end{array}$

[0128] where p is the specific resistance of electrolyte which fills the pore. The R.sub.tot represents the total resistance.

The Fabrication of Thrombin-Binding Aptamer (TBA) Modified AuNP Dimers

[0129] The 17 nm AuNPs were concentrated 10-fold and resuspended in 0.5×TBE buffer to a final concentration of 10 nM. The prepared AuNPs were then mixed with the ssDNA5 (10 μM, dissolved in TE buffer), in a DNA:AuNP molar ratio of 5:1. NaCl solution (5 M) was added to the mixture to a final NaCl concentration of 50 mM, and left at 25° C. for 12 h. Finally, the mixture was centrifuged (three times) at 7000 rpm for 10 min to remove the excess DNA, and resuspended in TBE buffer. The process of AuNPs (17 nm) modified with the ssDNA6 were same as DNAS. 100 μL of AuNP-DNAS and 100 μL of AuNP-DNA6 were mixed in TBE buffer containing 50 mM NaCl. The mixture was hybridised for 12 h at room temperature with gentle stirring. The mixture was centrifuged at 5000 rpm for 10 min to remove the single nanoparticles and collect the AuNPs dimer thrombin carrier. The sequence of DNA is shown in FIG. 6.

The Fabrication of the Monomer PCT Probes

[0130] 2 mL AuNPs (20±3 nm) were centrifuged for 10 min at 8000 rpm and then resuspended in 200 μL of 10 mM phosphate buffer (PB) solutions, which was adjusted to pH 9 with 0.1 M K2CO3. Next, 100 μL of the AuNPs were conjugated with anti-PCT mAb (10 μL, 100 μg/m L) and the other 100 μL AuNPs were modified with anti-PCT smAb (10 μL, 100 μg/mL), respectively. Then, the solutions were blocked by BSA (10 μL, 500 μg/mL) for 30 min after the incubation of 1 h. Next, the functionalized AuNPs solutions were centrifuged for 10 min at 7500 rpm so that the final product can be collected as the monomer PCT probes.

Nanostructures Translocation Experiment

[0131] The translocation experiments were performed from inside nanopipette to outside, where the AuNPs based nanostructures are loaded in the nanopipette which is the cis chamber as well as an Ag/AgCl working/patch electrode while an Ag/AgCl counter/reference electrode was placed in the blank buffer located in the bath as the trans chamber. The buffer used in the translocation experiments consisted of 50 mM KCl. 10 mM Tris-EDTA (pH=8) unless reported otherwise. For the binding assay of thrombin or PCT by using corresponding strategies, 1 nM thrombin carriers or PCT probes are incubated with target analytes at a different concentration at least 2 hrs. The buffer containing target analytes were filled inside the nanopipette along with the electrode. Then a voltage was applied by a high bandwidth amplifier VC100 (Chimera Instruments) between the electrodes on both sides of the nanopore, and the current-time trace can be recorded. The data was then resampled to 1 μs and refiltered to 100 kHz and was analysed by a customised Matlab App.

Results

Quantification of AuNP Monomers, Dimers, and Trimers

[0132] To demonstrate the screening ability of the AuNP conjugates, we quantified the resolution of our platform. First, we confirmed the ability to differentiate between monomers and dimers linked with differing DNA lengths. AuNP monomers were fabricated by attaching a thiol-modified 25-mer ssDNA to the surface of 17 nm in diameter AuNPs (FIG. 2a). AuNP symmetrical dimers were fabricated by self-assembly of two AuNP monomers with one consisting of a 15 bases complementary sequence. To challenge the spatial resolution, we fabricated two kinds of AuNP symmetrical dimers: one with 35 bases linkers and the other with 115 bases linkers. Asymmetrical dimers we also designed consisting of 10 nm AuNP monomers and 20 nm AuNPs. Finally, trimers were also quantified and could be achieved by controlling the NP to DNA ratio.

The Fabrication of AuNP Based Nanostructures

AuNP Monomer

[0133] The synthesised AuNPs (17 nm) were concentrated 10-fold and resuspended in 0.5×TBE buffer to a final concentration of 10 nM. The prepared AuNPs were then modified with the ssDNA1 (10 μM, dissolved in TE buffer), in a DNA:AuNP molar ratio of 5:1. NaCl solution (5 M) was added to the mixture to a final NaCl concentration of 50 mM, and left at 25° C. for 12 h. Finally, the mixture was centrifuged (three times) at 7000 rpm for 10 min to remove the excess DNA and resuspended in TBE buffer. The schematic of AuNP monomer is shown in FIG. 7a.

AuNP Symmetrical Dimer (35 Bases Linker)

[0134] The process of AuNPs (17 nm) modified with the ssDNA2 were same as DNA1. 100 μL of AuNP-DNA1 and 100 μL of AuNP-DNA2 were mixed together in TBE buffer containing 50 mM NaCl. The mixture was hybridized for 12 h at room temperature with gentle stirring. The mixture was centrifuged at 5000 rpm for 10 min to remove the single nanoparticles and collect the AuNP symmetrical dimers with short linker. The schematic of AuNP symmetrical dimer (35 bases linker) is shown in FIG. 7b.

AuNP Asymmetrical Dimer

[0135] AuNPs were concentrated 10-fold and resuspended in 0.5×TBE buffer to a final concentration of 10 nM. The prepared 20 nm AuNPs were then modified with the ssDNA1 (10 μM, dissolved in TE buffer, mixture1) and 10 nm AuNPs were then modified with the ssDNA2 (10 μM, dissolved in TE buffer, mixture2), in a DNA:AuNP molar ratio of 5:1. NaCl solution (5 M) was added to the mixture to a final NaCl concentration of 50 mM, and left at 25° C. for 12 h. Finally, the mixture1 was centrifuged (three times) at 7000 rpm for 10 min, and the mixture2 was centrifuged (three times) at 8500 rpm for 10 min to remove the excess DNA and resuspended in TBE buffer. 100 μl of 20 nm AuNP-DNA1 and 100 μl of 10 nm AuNP-DNA2 were mixed together in TBE buffer containing 50 mM NaCl. The mixture was hybridized for 12 h at room temperature with gentle stirring. The mixture was centrifuged at 5000 rpm for 10 min to remove the single nanoparticles and collect the AuNP asymmetrical dimers. The schematic of AuNP asymmetrical dimer is shown in FIG. 7c.

AuNP Trimer

[0136] 100 μl of 17 nm AuNP-DNA1 and 200 μL of 17 nm AuNP-DNA2 were mixed together in TBE buffer containing 50 mM NaCl. The mixture was hybridized for 12 h at room temperature with gentle stirring. The mixture was centrifuged at 4000 rpm for 10 min to remove the single nanoparticles and collect the AuNPs trimers. The schematic of AuNP trimer is shown in FIG. 7d.

AuNP Symmetrical Dimer (115 Bases Linker)

[0137] The 17 nm AuNPs were concentrated 10-fold and resuspended in 0.5×TBE buffer to a final concentration of 10 nM. The prepared AuNPs were then modified with the ssDNA3 (10 μM, dissolved in TE buffer), in a DNA:AuNP molar ratio of 5:1. NaCl solution (5 M) was added to the mixture to a final NaCl concentration of 50 mM, and left at 25° C. for 12 h. Finally, the mixture was centrifuged (three times) at 7000 rpm for 10 min to remove the excess DNA, and resuspended in TBE buffer. The process of AuNPs (17 nm) modified with the ssDNA4 were same as DNA3. 100 μL of AuNP-DNA3 and 100 μL of AuNP-DNA4 were mixed together in TBE buffer containing 50 mM NaCl. The mixture was hybridized for 12 h at room temperature with gentle stirring. The mixture was centrifuged at 5000 rpm for 10 min to remove the single nanoparticles and collect the AuNP symmetrical dimers with long linker. The schematic of AuNP symmetrical dimer (115 bases linker) is shown in FIG. 7e.

4-ATP Labelled AuNP Symmetrical Dimers (SERS Sample)

[0138] The 35 nm AuNPs were concentrated 10-fold and resuspended in 0.5×TBE buffer to a final concentration of 10 nM. The prepared AuNPs were then modified with the ssDNA1 (10 μM, dissolved in TE buffer), in a DNA:AuNP molar ratio of 5:1. NaCl solution (5 M) was added to the mixture to a final NaCl concentration of 50 mM, and left at 25° C. for 12 h. Finally, the mixture was centrifuged (three times) at 5000 rpm for 10 min to remove the excess DNA and resuspended in TBE buffer.

[0139] Before modified with the ssDNA2, 4-Aminothiophenol was added to the 35 nm AuNPs, with the final concentration of 10 μM. After 6 h, the AuNPs were centrifuged at 5000 rpm for 10 min to remove the unconnected 4-Aminothiophenol. Then, the above AuNPs were modified with ssDNA2. Finally, 100 μl of 35 nm AuNP-DNA1 and 100 μL of 35 nm AuNP-DNA2 were mixed together in TBE buffer containing 50 mM NaCl. The mixture was hybridized for 12 h at room temperature with gentle stirring. The mixture was centrifuged at 3000 rpm for 10 min to remove the single nanoparticles and collect the 4-ATP labelled AuNP symmetrical dimers. In order to increase the stability of the large AuNPs in high salt concentration, the SERS samples are further modified by PEG-SH. The schematic of 4-ATP labelled AuNP symmetrical dimers is shown in FIG. 7f.

[0140] The geometry of the nanostructures was confirmed by transmission electron microscopy (TEM), FIG. 2. To avoid the unpredictable orientation of the conjugated molecules with high viscosity during translocation, all AuNPs based samples were not protected by polyethylene glycol (PEG) unless otherwise stated. All NP conjugates were dispersed after fabrication and measured by UV-Vis after 24 hours stabilisation in the buffer 50 mM KCl, 10 mM Tris-EDTA and used in nanopore experiments. This ionic strength was chosen to minimise NP aggregation and at the same time maximise the signal to noise of the measurements.

[0141] Nanopore experiments were done using single-barrelled nanopipettes.sup.39, 40, which can be fabricated by laser-assisted pulling of quartz capillaries. The nanopores were pulled with an average diameter slightly larger than the dimensions of the nanoparticles, with and average diameter of 34±5 nm, as measured by scanning electron microscope (SEM). These dimensions closely matched the diameters estimated from nanopore conductance measurements ( ) of 15.3±2.4 nS in 100 mM KCl (n=18). Using SEM imaging, the taper angle of the nanopipette tip was measured to be 16.9±1.1° over the first 100 nm (n=18), which allowed us to estimate an effective sensing length between 25 to 50 nm, based on a 75-80% resistance drop at the nanopore. The analyte was filled inside the nanopipette where a patch electrode (AgCl) was placed, and a ground/reference electrode (AgCl) was placed in a bath, outside the pipette. By applying a negative potential, it was possible to transport the AuNPs from inside (cis) to outside (trans) of the nanopipette. A high bandwidth amplifier (Chimera VC100) was used with a 1 μs sampling rate and 100 kHz low-pass digital filter.

[0142] FIG. 2 shows a comparison of translocation characteristics between the different conjugates. The simplest constructs, AuNP monomers, translocated relatively quickly in a single file translocation with a with a single peak dwell time distribution (mean dwell time of 8.3±1.4 μs at an applied potential of −600 mV), FIG. 2a. In comparison symmetric and asymmetric dimers with a 35 base DNA spacer, FIG. 2b-2c exhibited clear double file events. The current amplitudes of each subpeak are consistent with the size of the individual AuNPs within the conjugate. The observations are consistent with the model that the first peak appears due to the negatively charged AuNPs passing though the nanopore sensing area. This is followed by a decrease of the current due to the reduced charge on the linker. Finally, the 2.sup.nd AuNP results in a 2.sup.nd peak. The mean dwell time of the dimers in FIG. 2b is 20.8±2.7 μs which is just over twice the monomer translocation time and is consistent with linear transport through the nanopore. Importantly, one could distinguish between the peaks with high spatial resolution, as the distance between the peaks was a mere 28 nm. Furthermore, it was possible to differentiate between spacers of different lengths, which correspond to distance of 28 and 51 nm, respectively.

[0143] When asymmetric AuNPs dimers were translocated out of the nanopipette (FIG. 2c), their asymmetric size was reflected in the shape of individual event in current time traces as well as the corresponding current peak distributions. The double peak current distributions were recorded with high peak currents of 155.51±21.55 pA and 81.83±13.47 pA, corresponding to translocations of 20 nm and 10 nm AuNP, respectively. Interestingly, nearly 95% of all translocation events of asymmetric dimers showed a preferential orientation with the larger NP being transported first, which was attributed to the larger NPs carrying higher surface charge. Although this was not the focus of the present manuscript, we also investigated the possibility of translocating and detecting NP trimers, FIG. 2d. Trimers could be detected with well-defined individual monomer signatures and it took 32.7±5.0 μs for individual trimers to translocate through the nanopore, which is 3.94-folds (4.23-folds for spatial length) longer than the monomer dwell time and 1.57-folds (1.61-folds for spatial length) longer than the dimer dwell time, respectively.

[0144] To further characterise the translocations of these metallic nanoparticles, single particle surface-enhanced Raman scattering (SERS) was also performed on a modified gold coated (10 nm thickness) nanopipette. Due to the coupling and proximity between dimer and metal pipette surface, this results in a significant increase in Raman signal..sup.41, 42 To achieve single-particle SERS somewhat larger 35 nm AuNP symmetrical dimers we used due to the higher scattering cross-section. The AuNPs were functionalized with 4-Aminothiophenol (ATP) dye (FIG. 2e, FIG. 7). The dimer is further stabilized with PEG to ensure the particles do not aggregate at the 100 mM salt concentrations required to perform the translocations. A typical SERS spectrum of the NPs in bulk solution is shown and consists of expected peaks at 1138, 1387 and 1571 cm.sup.−1 (FIG. 8). This is comparable to the data obtained for single particle SERS as can be seen from the transients in FIG. 2e. The integration time for the transients was 810 μs, and translocations were obtained at −800 mV. In this example, the optical translocation times were 3.24 ms which is longer than the corresponding electrical events (0.79±0.29 ms). This is due to the nanopore sensing region being much smaller than the diffraction limited laser spot size (ca. 1 μm). As a negative control, Raman spectra, which shows no Raman signal, were also acquired for the event when the reverse potential is applied to diffuse the dimer away from the nanopore. We envisage that this method can also be used to perform molecular assays and complements the electrical work shown in this manuscript.

The Design of AuNPs Dimer Thrombin Carrier and the Sensing of it with Thrombin

[0145] The first strategy for the biosensing is to utilise the dimer molecule as a carrier to drive and detect specific protein molecules. The AuNP dimer protein carrier, which is based on AuNP symmetrical dumbbell system with a DNA bridge, was engineered to contain a part of ssDNA overhang with the specific aptamer sequence. Due to the high affinity and selectivity of the aptamer and protein interaction, the dimer protein carriers with specific aptamer will only bind corresponding proteins in trace level, FIG. 3a.

[0146] In this case, human alpha-thrombin (α-thrombin; M.W. 37.5 kDa; pI of 7.0-7.4), a multifunctional protease in the bloodstream, became our target due to its significant roles in various crucial physiological and pathological processes, such as blood coagulation, thrombosis and angiogenesis. It is essential to detect thrombin at a trace amount with high sensitivity. However, using plain solid-state nanopores to sense this biomarker with such small size and heterogeneous charge, is difficult to achieve. Therefore, AuNPs dimer thrombin carrier was designed to pave the way of thrombin detection at the single-molecule level.

[0147] The thrombin-binding aptamer (TBA), which binds to thrombin selectively and compactly (K.sub.d.sup.˜35-100 nM in solid phase assays.sup.36), is a 15-mer (5′-GGTTGGTGTGGTTGG-3′—SEQ ID NO: 10) ssDNA folding into stable intramolecular G-quadruplex in the presence of K.sup.+..sup.43, 44 The TBA sequence was anchored on a protuberance of the dimer 115-mer DNA bridges as a binding site of thrombin located in the middle of the dumbbell molecules, FIG. 6. Prior to nanopore measurements, the efficiency of the binding between thrombin and the corresponding carriers was confirmed by UV-vis. By adding 4 nM thrombin to 1 nM, AuNP dimer thrombin carrier dispersions with 50 mM KCl and 10 mM Tris-EDTA at pH 8, the AuNPs based molecules started oligomerisation due to the selective binding weakening the holistic charge of the molecule. As a control, the pristine AuNPs dimer molecules in the same ionic strength would not aggregate until the concentration of thrombin reached 100 nM, which masked the surface of the dimer molecules.

[0148] As a control, we first examine the translocation of TBA modified AuNP dimer, FIG. 3b. Comparing the translocation events of unmodified AuNPs dimer (FIG. 10) and TBA modified AuNPs dimer (FIG. 3b) with same particle size and same linker length, the difference in dwell time, peak current, and fraction position are negligible. With the addition of 1 nM thrombin into 1 nM TBA modified AuNPs dimer, some triple peak events were observed when these complexes were driven by −600 mV potential, FIG. 3c. Unlike the triple peaks of the AuNPs trimers, the triple peaks events of the protein bound dimer revealed a distinct signature that the second peak (located on 0.51 of the normalised peak position) always smaller than the first and third peak (located on 0.22 and 0.86 of the normalised peak position, respectively). As discussed before, the peaks are generated when the nanopore conductance changed by the different part of the nanostructures. In the pH 8 environment, the thrombin and TBA part are negatively charged which is ascribable to the deprotonation of the amino acid, although it is not comparable with the charge of the AuNPs. Therefore, the small enhancement peak, with an 80% magnitude of the AuNPs peaks, occurred at the middle of the events, corresponding to the thrombin anchored DNA bridges. However, proved by the UV-Vis, the holistic charge of the molecular carrier is weakened after the addition of thrombin, which leading a 1.1-fold increase of the translocation dwell time, FIG. 10.

[0149] By counting the percentage of the triple peak events of all triple peak and double peak events, the binding ratio can be calculated, FIG. 3d-e. As expected, the proportion of triple peak signature raised with the addition of thrombin. In this experiment, we varied the concentration of thrombin from 0 to 2 nM while the concentration of the dimer remained 1 nM. From the statistics of normalised peak position (FIG. 3d), it is evident that there are no triple peak events without the addition of thrombin and then the middle peak showed stepwise with the increasing of the thrombin concentration. The subtle change of the concentration of thrombin can be monitored as approximate 50 pM change of the thrombin; the triple/double peak ratio will change 1%. Further, to validate the selectivity of the AuNP dimer thrombin carrier, 1 nM lysozyme was incubated with 1 nM thrombin carrier and subsequent nanopore detection of the mixture was performed, FIG. 11. There is no sign of triple peak signature but the double peak events, which are corresponding to the unbounded dimer carrier.

The Design of AuNP Monomer PCT Probes and the Detection of PCT in Single Molecule Level

[0150] The AuNP dimer protein carriers showed the ability to sense corresponding protein with high sensitivity and selectivity. However, the aptamers modified carriers, including most carriers reported previously, cannot sense smaller targets (<15 kDa). What is worse, with the decreasing of the biomolecule size, the signatures become progressively hard to detect due to the lowering of the signal-to-noise ratio.

[0151] Herein, based on our previous work.sup.45,46, a universal strategy of sensing small antigen molecules has been performed. In detail, half of AuNPs are modified by the corresponding antibody (mAb) of the target antigen while the rest are modified with complementary secondary antibody (smAb). With the presence of the antigen, the monomers will self-assemble to dimer with a ‘sandwich’ formation (FIG. 4a) and this changing can be recorded when the molecules pass through the nanopore.

[0152] In this case, we use the AuNP monomer antigen probes to detect procalcitonin (PCT; M.W. 12.8 kDa; pI of 6.5) which is a peptide precursor of the hormone calcitonin. Due to the PCT level variance between healthy and microbial infected individuals, it has become an important biomarker to improve bacterial infections identification and guide antibiotic therapy. A pair of AuNP monomer PCT probes were fabricated to sensing PCT in single-molecule level, which cannot be studied by the conventional translocation due to the extremely small size. To ensure the intramolecular nanostructure can be distinguished by the nanopore in this case, we increased the AuNP size to 20 nm. Therefore, with an antibody-antigen-antibody sandwich linker which is approximately 10 nm, the substructure of the dumbbell molecule (the centre of one AuNP to the other is around 30 nm) can be distinguished by nanopores with sub-30 nm effective length.

[0153] A comparison between the translocation of 2 nM AuNP monomers (50% are functionalized by PCT mAb, and 50% are functionalized by smAb) and the assembled dumbbell complex after adding 20 nM PCT, was shown in FIG. 4b-c. Without the presence of PCT, no double peak signal but a single peak signal was observed during the translocation of 2 nM AuNP monomers with 50 mM KCl, pH 8. With the addition of 20 nM PCT, about 15% of the single peaks transformed to clear double peak events. Each peak is the result of the translocating of AuNP whereas the trough is due to the translocation of the weak and uniform charged sandwich linker (PI of mAb, smAb is 6.6-7.2). As a negative control, no double peak signatures observed when PCT was replaced by other antigens, for example, insulin, FIG. 12.

[0154] To validate the sandwich immunoassay mode can be used in clinical diagnosis level, the nanopore sensing studies of AuNP monomer probes with different concentration of PCT was performed. In this case, the concentration of the probes was kept as 2 nM while the concentration of PCT was ranging from 0 to 200 nM. As expected, the percentage of the single peak (located on 0.49 of the normalised peak position) decreased whereas the proportion of double peak signatures (located on 0.30 and 0.78 of the normalised peak position) raised with the increasing of PCT, FIG. 4d(i). The results were further confirmed by TEM (FIG. 4d(ii)), which provided visualised evidence that the proportion of dimer increased with more addition of PCT.

[0155] In bacterial infections, sepsis, severe sepsis and septic shock, PCT in plasma concentrations increases from 0.15 to more than 10 ng/ml. This increase often correlates with the severity of the disease and with mortality. At the same time, PCT has also been used to guide antibiotic therapy, for example, if PCT level <0.1 ng/ml, antibiotic therapy is strongly discouraged; if PCT level >1 ng/ml, antibiotic therapy is strongly encouraged. With high sensitivity, the PCT monomer probe is capable of sensing this important biomarker in this range (FIG. 4e inset).

Molecular Probes for Single-Molecule Detection of miRNA

[0156] miRNA are a class of short non-coding RNAs that function in RNA silencing and post-transcriptional gene regulation. Besides their participation in regulating normal physiological activities, specific miRNA types could act as oncogenes, tumor suppressors, or metastasis regulators, which are critical biomarkers for cancer. Conventional methods include Northern blotting, in situ hybridization, RT-qRCR, or microarrays. However, these methods require sample preparation or processing. In addition each technique has specific limitations such as low throughput and low sensitivity (for northern blotting), semi-quantitative (for in situ hybridization), time consuming, critical reaction condition (for RT-qPCR). Recent advances in nanopore technology offer the promise of addressing some of these drawbacks for detection of miRNA with high sensitivity and selectivity.[47] However, the signal of these short fragments (typically 18-23 bases) is hard to detect directly with solid-state nanopores due to the high-speed translocation and low signal-to-noise ratio, FIG. 16. Here, we use AuNP dimer self-assembly to amplify this translocation signal, leading to very efficient miRNA detection at the single-molecule level.

[0157] In this study, we use AuNP molecular probes for the detection of miRNA-141. miRNA-141 is commonly dysregulated in malignant tumors such as those associated with prostate cancer and plays essential roles in tumor development and progression, becoming a powerful potential biomarker of prostate cancer.[48] Prostate cancer is the second most common cancer in men worldwide; however, disease outcome is difficult to predict in large part due to the lack of efficient diagnostic strategies. As such, miRNA-141 has the potential to become a useful biomarker.

[0158] The molecular probes consisted of two populations of ssDNA functionalized to AuNP monomers. Each of them was modified by an 11 base recognition chain, which can hybridize with half of the 22-base-long miRNA-141, FIG. 17. With the addition of the target, the monomer probes self-assemble to form dimers and produce doublet signatures, FIG. 15a-c. A binding assay was performed within the miRNA concentration range of 1 pM to 100 nM. As previously shown for PCT, the number of dimers, and hence doublets, increases with concentration, FIG. 15d-e. Dimer formation is validated and compared with TEM, FIG. 18, providing visual evidence of dimer formation due to the presence of miRNA-141. Typically, the concentration of miRNA-141 between fM and pM in unprocessed prostate cancer patient samples, and between pM to nM in extracted miRNA samples.

[0159] The specificity of the molecular probes was verified by detecting miRNA-200a, which is also in the miR-200 family and share seed sequences differing in only two nucleotides when compared with miRNA-141, FIG. 19. The full recognition of miRNA-141 gives a significant binding result, whereas the control experiment, which is detecting the miRNA-200a, leads to a low value of the binding ratio, FIG. 15e. The result is further confirmed by TEM, FIG. 15e, FIG. 20. Such high selective capability probably benefits from the dimerization mechanism. For example, for miRNA-141, the monomer probes can be linked to the dimer because the ssDNA is fully matching the target. In contrast, for the miRNA-200a, the two mismatch points happened on the same ssDNA of one monomer probe, leading to a very low binding affinity, which causes unsuccessful dimerization. This result shows that the AuNP monomer probe can detect the target with high specificity.

CONCLUSION

[0160] Although nanoparticle-based superstructures have already been reported and some of them are detected by nanopore sensing49, many of these tests are based on sensing the exclude volume rather than the substructures of these nanoparticle conjugates, leading to false positive in the nanoparticle based sensing applications. We demonstrate that it is possible to use a finely tuned nanopore testing platform incorporating with high-bandwidth instruments to depict the sub-structure of these molecules. We have shown this set-up can differentiate AuNP monomer, AuNPs symmetrical or asymmetrical dimer, and AuNPs trimer. By utilising the plasmonic effect of the dimer system, the single molecule SERS detection was also applied. Based on these validations of the detection resolution, two strategies of sensing biomolecules in a single molecule level are performed.

[0161] In summary, the first strategy is to use an AuNPs dimer protein carrier, which is an AuNPs dumbbell system incorporating with an aptamer prominence on the middle of the DNA linker, to detect proteins. The second strategy is to utilise the feature of the self-assembling of AuNP monomer antigen probes to dimers with the addition of the specific antigens. Both strategies are fully flexible to detect a number of biomolecules with just changing the aptamer sequence or antibodies. The excellent selectivity and affinity of aptamer-protein or antibody-antigen provide the possibility to apply these strategies to diagnostics for detecting biomarkers in trace amount. Importantly, to different biomolecules, different strategy can be chosen. For example, to sense some large proteins, the aptamer modified carrier can be used because an evident signature in the middle can provide information such as the size and charge of the target. Otherwise, if the biomolecule is too small to generate the ripples on the electric signal, the other strategy, an indirect detection in single molecule level, can be applied. Both strategies are not only capable of validating the presence of the specific targets, but also can quantify the concentration of them in clinical diagnosis level.

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