Patent Description:
Early and rapid diagnosis of disease is an essential step in enhancing effectiveness of treatments and in reducing morbidity and mortality. As a non-invasive and cost-effective tool for early diagnosis and prognosis of acute and chronic diseases, the detection of biomarkers, especially at a single-molecule level, have attracted researchers' attention for decades.

Meanwhile, the exquisite design of micro/nano environmental sensing methods and devices allows the detection of analytes at a single molecule level. With size-controllable features, nanopores<NUM>, <NUM>, <NUM>, <NUM>, <NUM> have been proven to have the capability of detecting single DNA<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, protein<NUM>, <NUM>, <NUM>, nanoparticles<NUM>, <NUM>, <NUM>, <NUM>, <NUM> and other molecules<NUM>, <NUM>, <NUM>. The detection method is straightforward; Targeting molecules are translocated through a nanometer-sized pore using an applied electric field, and the translocations are detected by the transient changes of ionic current. The magnitude and dwell time of the current change provides information on the structural features of the analytes. However, the screening of small biomolecules using nanopore remained remarkably challenging due to their small size, heterogeneous charge, fast translocation and low signal-to-noise ratio.

Attempts have been made to address some of these limitations by functionalizing the nanopore surface with hydrophobic, and positively or negatively charged binding sites<NUM>, <NUM>, <NUM>. In addition, aptamer encoded glass nanopores<NUM>, <NUM>, chemically modified nanopores with binding sites<NUM>, <NUM>, <NUM>, and nanopipette based field-effect-transistors<NUM>, <NUM>, <NUM> have also been investigated. However, functionalizing nanopores is often challenging due to complicated fabrication processes and the requirement for careful optimization. As another potential solution, high bandwidth amplifiers have been used to detect small molecules<NUM>, <NUM>. Unfortunately, these high-resolution instruments are not capable of differentiating multiple kinds of biomolecules. Therefore, fundamental challenges remain in the sensitive and selective detection of biomarkers such as micro RNA, proteins and small antigen molecules using label-free solid state nanopores.

An alternative solution involves the use of molecular carriers, which include specific sites that interact with the molecules they transport. Singer et al. pioneered the concept of utilising the DNA as molecular carriers<NUM>. Bell et al. used nanopipettes and a carrier based on a <NUM> kbp DNA including a sequence of structural features (dumbbells) as a 'barcode' identifying the DNA and a binding site which can interact with targeting antibodies selectively<NUM>. Recently, Sze et al. designed a delicate multiple proteins carrier based on λ-DNA with ssDNA overhang which can anchor aptamers to bind targeting proteins (up to three) selectively<NUM> and 35a. Combining the above concepts, more sophisticated molecular carriers have been reported<NUM>, <NUM>. However, these designs are restricted by many factors such as the relatively high concentration of targeting analytes, especially proteins, and high ionic strength of buffer/sample required to prevent non-specific binding due to the shielding effect provided by salt ions. Moreover, the folding of long-chain DNA based carrier backbone<NUM> can cause interference in the identification of the analyte signal, while the short carriers are hard to fabricate, purify and embed the binding sites. These approaches are also not capable of sensing small molecules (<15kDa) due to the poor signal-to-noise ratios. <CIT> is directed to a target molecule modified to facilitate detection in a nanopore device, and a method of detecting such a modified target molecule using a nanopore device. <NPL>) describes selective single molecule nanopore sensing of proteins using DNA aptamer-functionalised gold nanoparticles. <NPL>) describes a rapid and label-free single-nucleotide discrimination via an integrative nanoparticle-nanopore approach. <CIT> describes nucleic acid nanoparticles for analyte detection. <NPL>) describes direct detection of antibody-antigen binding using an on-chip artificial pore. <CIT> describes the use of resistive-pulse sensing with submicrometer pores or nanopores for the detection of the assembly of submicrometer or nanometer sized objects.

To address the limitations of the prior art, the present invention provides sensing strategies which aim to increase the sensitivity and selectivity of nanopore biosensing by increasing the size of the analyte, in order to improve the signal-to-noise ratio. The scope of the present invention is set out in the appended set of claims.

Accordingly, in a first aspect there is provided a method of detecting one or more analytes in a target sample, the method comprising:.

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; 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; and wherein the analyte is a protein.

The nanoparticle dimer may be adapted to bind the analyte in a number of different ways.

Preferably, the nanoparticles may be linked by partially complementary nucleic acids.

Preferably, 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.

Preferably, the nanoparticles in the dimer are of substantially the same diameter. Alternatively, the nanoparticles in the dimer may be of different diameters.

Preferably, the nanopore is the tip of a nanopipette.

Preferably, the nanoparticles are gold nanoparticles (AuNPs).

Preferably, 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.

The biological sample may alternatively be any other bodily fluid or secretion in a state of health or disease.

The present inventors have exemplified the first aspect of the invention using a first strategy and two other strategies are described. The Examples of the present application relate to gold nanoparticles (AuNPs), but it will be appreciated that the 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.

The first strategy - for transporting and identifying relatively bigger single proteins (> <NUM> kDa) - implemented by a carrier which is a dumbbell system comprising two nanoparticles with an aptamer located at the middle of a DNA linker (<FIG>). Each end of the linker is attached to one of the nanoparticles. Aptamers are ssDNA or RNA oligonucleotides that are capable of binding target molecules with high specificity and affinity, where the sequence can be selected by SELEX (systematic evolution of ligands by exponential enrichment). The specific aptamer modified dimer carriers thus bind to target proteins selectively. Then the sub-structure of protein/carrier complexes can be identified when they pass through the nanopore along with high-resolution translocation signals. A specific example of such a configuration using AuNPs and ssDNA linkers is shown in <FIG>.

A second strategy is described, which is for detecting tiny antigen molecules (< <NUM> kDa) and is achieved by a pair of monomer (nanoparticle) probes, one being conjugated to an antibody to a target antigen and the other being conjugated to a complementary secondary antibody (<FIG>). The monomer (nanoparticle) probes will self-assemble to dimer molecules by the linkage of the antibody-antigen-antibody sandwich bridge after the addition of the specific antigen. In this strategy, the method of the first aspect of the invention can also be seen to include a step of causing the nanoparticles to form a dimer.

The two strategies can be combined (<FIG>): one nanoparticle is functionalised with ssDNA with a part of aptamer sequence at the end while the other nanoparticle is functionalised with ssDNA that includes the rest of aptamer sequence; when the target protein is present, the two aptamer parts will link together to form an aptamer which binds to the protein, leading to the conjugation of the nanoparticle monomers. It can thus be seen that in this embodiment the dimer is formed by formation of the aptamer. In this embodiment, the method of the first aspect of the invention can also be seen to include a step of causing the nanoparticles to form a dimer.

A third strategy is described, which can be used for the selective detection of nucleic acid molecules such as miRNA (<FIG>). Two populations of nanoparticles can be modified to each contain half (or at least two different portions) of a sequence complementary to an miRNA sequence. In the presence of miRNA, the monomeric nanoparticles will self-assemble and dimerize.

In all strategies, the translocation signal of the nanoparticle dimer compared to individual nanoparticles indicates the presence of the analyte. This allows for very accurate detection of analytes based on the peak shape. All strategies can be adapted to different targets (including small molecules such as small peptides and proteins) by changing the aptamer sequence or antibody to the required target. In addition, these strategies can be adapted to quantify concentration of the target. The strategies also allow detection of analytes in complex media as the analytes are not detected directly but rather via the formation of dimers.

The present inventors have also devised a strategy for using nanopore monomers for sensing analytes using functionalised nanoparticle probes and DNA carriers by biological nanopores. These aspects of the invention are shown in <FIG> and <FIG>, which illustrate the methods for AuNP probes.

The sequences referred to in the Figures are as follows:.

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: <NUM>) 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. <NUM>) Lack of single molecule data. This may cause the unspecific detection or recognise all isoforms of one same protein in a sample. <NUM>) Other factors such as complex sample preparation and time consumption. Therefore, a fast, accurate and specific single molecule detection method is urgently needed.

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.

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.

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.

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.

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 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.

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.

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 (<NPL>) and examples of these are given in the table below.

Abbreviations used in the table are as follows:.

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.

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.

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

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 <NUM>, and typically the data is filtered with <NUM> low pass filter.

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.

In one case, the nanoparticle monomer was based on AuNPs with <NUM>-<NUM> diameter with the surface functionalized by single stranded DNA (<NUM>-<NUM> bases). However, nanoparticles of a variety of sizes may be used in the present invention. An AuNP dimer is <NUM> 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 <NUM> (<NUM> bases) and a ssDNA <NUM> (<NUM> bases, DNA <NUM> is complementary with the end of DNA <NUM>). The aptamer with DNA <NUM> (<NUM>-<NUM> bases, DNA <NUM> is complementary with the unpaired part of DNA <NUM>) then attached to the dimer. The resulting AuNPs dimer with a branch of aptamer can be the molecular carrier of a specific protein.

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.

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.

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. 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.

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

The single barrel quartz capillaries (o. , <NUM>, i. , <NUM>, Intracell) were plasma cleaned (Harrick Plasma), and pulled with a laser-based P-<NUM> pipette puller (Sutter Instruments) using a two-line program (heat <NUM>, filament <NUM>, velocity <NUM>, delay <NUM>, and pull <NUM>; heat <NUM>, filament <NUM>, velocity <NUM>, delay <NUM>, and pull <NUM>) to produce nanopipettes with the nanopore diameters of approximately <NUM> 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 effective length of a nanopipette is the portion of the electrolyte-filled pore over which the majority (for our calculations <NUM>-<NUM>%) 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.

To detect the nanopipette effective length, the resistance distribution along the pore axis should be analysed (<FIG>). The resistance with different position (Rx) of the nanopore can be estimated. The distance from the end, x, is ranging from <NUM> 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.

Dx can be expressed as the following function of x: <MAT>.

In our calculation, the step of x is <NUM>.

The resistance Rx can be calculated as following function: <MAT> where ρ is the specific resistance of electrolyte which fills the pore. The Rtot represents the total resistance.

The <NUM> AuNPs were concentrated <NUM>-fold and resuspended in <NUM>×TBE buffer to a final concentration of <NUM>. The prepared AuNPs were then mixed with the ssDNA5 (<NUM>, dissolved in TE buffer), in a DNA: AuNP molar ratio of <NUM>:<NUM>. NaCl solution (<NUM>) was added to the mixture to a final NaCl concentration of <NUM>, and left at <NUM> for <NUM>. Finally, the mixture was centrifuged (three times) at <NUM> rpm for <NUM> to remove the excess DNA, and resuspended in TBE buffer. The process of AuNPs (<NUM>) modified with the ssDNA6 were same as DNA5. <NUM>µL of AuNP-DNA5 and <NUM>µL of AuNP-DNA6 were mixed in TBE buffer containing <NUM> NaCl. The mixture was hybridised for <NUM> at room temperature with gentle stirring. The mixture was centrifuged at <NUM> rpm for <NUM> to remove the single nanoparticles and collect the AuNPs dimer thrombin carrier. The sequence of DNA is shown in <FIG>.

<NUM> AuNPs (<NUM> ± <NUM>) were centrifuged for <NUM> at <NUM> rpm and then resuspended in <NUM>µL of <NUM> phosphate buffer (PB) solutions, which was adjusted to pH <NUM> with <NUM> K2CO3. Next, <NUM>µL of the AuNPs were conjugated with anti-PCT mAb (<NUM>µL, <NUM>µg/mL) and the other <NUM>µL AuNPs were modified with anti-PCT smAb (<NUM>µL, <NUM>µg/mL), respectively. Then, the solutions were blocked by BSA (<NUM>µL, <NUM>µg/mL) for <NUM> after the incubation of <NUM>. Next, the functionalized AuNPs solutions were centrifuged for <NUM> at <NUM> rpm so that the final product can be collected as the monomer PCT probes.

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 <NUM> KCl. <NUM> Tris-EDTA (pH=<NUM>) unless reported otherwise. For the binding assay of thrombin or PCT by using corresponding strategies, <NUM> thrombin carriers or PCT probes are incubated with target analytes at a different concentration at least <NUM> 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 <NUM> and refiltered to <NUM> and was analysed by a customised Matlab App.

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 <NUM>-mer ssDNA to the surface of <NUM> in diameter AuNPs (<FIG>). AuNP symmetrical dimers were fabricated by self-assembly of two AuNP monomers with one consisting of a <NUM> bases complementary sequence. To challenge the spatial resolution, we fabricated two kinds of AuNP symmetrical dimers: one with <NUM> bases linkers and the other with <NUM> bases linkers. Asymmetrical dimers we also designed consisting of <NUM> AuNP monomers and <NUM> AuNPs. Finally, trimers were also quantified and could be achieved by controlling the NP to DNA ratio.

The synthesised AuNPs (<NUM>) were concentrated <NUM>-fold and resuspended in <NUM>×TBE buffer to a final concentration of <NUM>. The prepared AuNPs were then modified with the ssDNA1 (<NUM>, dissolved in TE buffer), in a DNA: AuNP molar ratio of <NUM>:<NUM>. NaCl solution (<NUM>) was added to the mixture to a final NaCl concentration of <NUM>, and left at <NUM> for <NUM>. Finally, the mixture was centrifuged (three times) at <NUM> rpm for <NUM> to remove the excess DNA and resuspended in TBE buffer. The schematic of AuNP monomer is shown in <FIG>.

The process of AuNPs (<NUM>) modified with the ssDNA2 were same as DNA1. <NUM>µL of AuNP-DNA1 and <NUM>µL of AuNP-DNA2 were mixed together in TBE buffer containing <NUM> NaCl. The mixture was hybridized for <NUM> at room temperature with gentle stirring. The mixture was centrifuged at <NUM> rpm for <NUM> to remove the single nanoparticles and collect the AuNP symmetrical dimers with short linker. The schematic of AuNP symmetrical dimer (<NUM> bases linker) is shown in <FIG>.

AuNPs were concentrated <NUM>-fold and resuspended in <NUM>×TBE buffer to a final concentration of <NUM>. The prepared <NUM> AuNPs were then modified with the ssDNA1 (<NUM>, dissolved in TE buffer, mixture1) and <NUM> AuNPs were then modified with the ssDNA2 (<NUM>, dissolved in TE buffer, mixture2), in a DNA: AuNP molar ratio of <NUM>:<NUM>. NaCl solution (<NUM>) was added to the mixture to a final NaCl concentration of <NUM>, and left at <NUM> for <NUM>. Finally, the mixture1 was centrifuged (three times) at <NUM> rpm for <NUM>, and the mixture2 was centrifuged (three times) at <NUM> rpm for <NUM> to remove the excess DNA and resuspended in TBE buffer. <NUM>µL of <NUM> AuNP-DNA1 and <NUM>µL of <NUM> AuNP-DNA2 were mixed together in TBE buffer containing <NUM> NaCl. The mixture was hybridized for <NUM> at room temperature with gentle stirring. The mixture was centrifuged at <NUM> rpm for <NUM> to remove the single nanoparticles and collect the AuNP asymmetrical dimers. The schematic of AuNP asymmetrical dimer is shown in <FIG>.

<NUM>µL of <NUM> AuNP-DNA1 and <NUM>µL of <NUM> AuNP-DNA2 were mixed together in TBE buffer containing <NUM> NaCl. The mixture was hybridized for <NUM> at room temperature with gentle stirring. The mixture was centrifuged at <NUM> rpm for <NUM> to remove the single nanoparticles and collect the AuNPs trimers. The schematic of AuNP trimer is shown in <FIG>.

The <NUM> AuNPs were concentrated <NUM>-fold and resuspended in <NUM>×TBE buffer to a final concentration of <NUM>. The prepared AuNPs were then modified with the ssDNA3 (<NUM>, dissolved in TE buffer), in a DNA: AuNP molar ratio of <NUM>:<NUM>. NaCl solution (<NUM>) was added to the mixture to a final NaCl concentration of <NUM>, and left at <NUM> for <NUM>. Finally, the mixture was centrifuged (three times) at <NUM> rpm for <NUM> to remove the excess DNA, and resuspended in TBE buffer. The process of AuNPs (<NUM>) modified with the ssDNA4 were same as DNA3. <NUM>µL of AuNP-DNA3 and <NUM>µL of AuNP-DNA4 were mixed together in TBE buffer containing <NUM> NaCl. The mixture was hybridized for <NUM> at room temperature with gentle stirring. The mixture was centrifuged at <NUM> rpm for <NUM> to remove the single nanoparticles and collect the AuNP symmetrical dimers with long linker. The schematic of AuNP symmetrical dimer (<NUM> bases linker) is shown in <FIG>.

The <NUM> AuNPs were concentrated <NUM>-fold and resuspended in <NUM>×TBE buffer to a final concentration of <NUM>. The prepared AuNPs were then modified with the ssDNA1 (<NUM>, dissolved in TE buffer), in a DNA: AuNP molar ratio of <NUM>:<NUM>. NaCl solution (<NUM>) was added to the mixture to a final NaCl concentration of <NUM>, and left at <NUM> for <NUM>. Finally, the mixture was centrifuged (three times) at <NUM> rpm for <NUM> to remove the excess DNA and resuspended in TBE buffer.

Before modified with the ssDNA2, <NUM>-Aminothiophenol was added to the <NUM> AuNPs, with the final concentration of <NUM>. After <NUM>, the AuNPs were centrifuged at <NUM> rpm for <NUM> to remove the unconnected <NUM>-Aminothiophenol. Then, the above AuNPs were modified with ssDNA2. Finally, <NUM>µL of <NUM> AuNP-DNA1 and <NUM>µL of <NUM> AuNP-DNA2 were mixed together in TBE buffer containing <NUM> NaCl. The mixture was hybridized for <NUM> at room temperature with gentle stirring. The mixture was centrifuged at <NUM> rpm for <NUM> to remove the single nanoparticles and collect the <NUM>-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 <NUM>-ATP labelled AuNP symmetrical dimers is shown in <FIG>.

The geometry of the nanostructures was confirmed by transmission electron microscopy (TEM), <FIG>. 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 <NUM> hours stabilisation in the buffer <NUM> KCI, <NUM> 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.

Nanopore experiments were done using single-barrelled nanopipettes<NUM>, <NUM>, 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 <NUM> ± <NUM>, as measured by scanning electron microscope (SEM). These dimensions closely matched the diameters estimated from nanopore conductance measurements () of <NUM> ± <NUM> nS in <NUM> KCI (n = <NUM>). Using SEM imaging, the taper angle of the nanopipette tip was measured to be <NUM> ± <NUM>° over the first <NUM> (n=<NUM>), which allowed us to estimate an effective sensing length between <NUM> to <NUM>, based on a <NUM>-<NUM>% 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 <NUM> sampling rate and <NUM> low-pass digital filter.

<FIG> 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 <NUM> ± <NUM> at an applied potential of -<NUM> mV), <FIG>. In comparison symmetric and asymmetric dimers with a <NUM> base DNA spacer, <FIG> 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 <NUM>nd AuNP results in a <NUM>nd peak. The mean dwell time of the dimers in <FIG> is <NUM> ± <NUM> 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 <NUM>. Furthermore, it was possible to differentiate between spacers of different lengths, which correspond to distance of <NUM> and <NUM>, respectively.

When asymmetric AuNPs dimers were translocated out of the nanopipette (<FIG>), 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 <NUM> ± <NUM> pA and <NUM> ± <NUM> pA, corresponding to translocations of <NUM> and <NUM> AuNP, respectively. Interestingly, nearly <NUM>% 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>. Trimers could be detected with well-defined individual monomer signatures and it took <NUM> ± <NUM> for individual trimers to translocate through the nanopore, which is <NUM>-folds (<NUM>-folds for spatial length) longer than the monomer dwell time and <NUM>-folds (<NUM>- folds for spatial length) longer than the dimer dwell time, respectively.

To further characterise the translocations of these metallic nanoparticles, single particle surface-enhanced Raman scattering (SERS) was also performed on a modified gold coated (<NUM> thickness) nanopipette. Due to the coupling and proximity between dimer and metal pipette surface, this results in a significant increase in Raman signal. <NUM>, <NUM> To achieve single-particle SERS somewhat larger <NUM> AuNP symmetrical dimers we used due to the higher scattering cross-section. The AuNPs were functionalized with <NUM>-Aminothiophenol (ATP) dye (<FIG>, <FIG>). The dimer is further stabilized with PEG to ensure the particles do not aggregate at the <NUM> 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 <NUM>, <NUM> and <NUM>-<NUM> (<FIG>). This is comparable to the data obtained for single particle SERS as can be seen from the transients in <FIG>. The integration time for the transients was <NUM>, and translocations were obtained at - <NUM> mV. In this example, the optical translocation times were <NUM> which is longer than the corresponding electrical events (<NUM> ± <NUM>). This is due to the nanopore sensing region being much smaller than the diffraction limited laser spot size (ca. 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 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>.

In this case, human alpha-thrombin (α-thrombin; M. <NUM> kDa; pl of <NUM>-<NUM>), 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.

The thrombin-binding aptamer (TBA), which binds to thrombin selectively and compactly (Kd ~ <NUM>-<NUM> in solid phase assays<NUM>), is a <NUM>-mer (<NUM>'-GGTTGGTGTGGTTGG-<NUM>' - SEQ ID NO: <NUM>) ssDNA folding into stable intramolecular G-quadruplex in the presence of K+. <NUM>, <NUM> The TBA sequence was anchored on a protuberance of the dimer <NUM>-mer DNA bridges as a binding site of thrombin located in the middle of the dumbbell molecules, <FIG>. Prior to nanopore measurements, the efficiency of the binding between thrombin and the corresponding carriers was confirmed by UV-vis. By adding <NUM> thrombin to <NUM>, AuNP dimer thrombin carrier dispersions with <NUM> KCl and <NUM> Tris-EDTA at pH <NUM>, 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 <NUM>, which masked the surface of the dimer molecules.

As a control, we first examine the translocation of TBA modified AuNP dimer, <FIG>. Comparing the translocation events of unmodified AuNPs dimer (<FIG>) and TBA modified AuNPs dimer (<FIG>) with same particle size and same linker length, the difference in dwell time, peak current, and fraction position are negligible. With the addition of <NUM> thrombin into <NUM> TBA modified AuNPs dimer, some triple peak events were observed when these complexes were driven by -<NUM> mV potential, <FIG>. 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 <NUM> of the normalised peak position) always smaller than the first and third peak (located on <NUM> and <NUM> 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 <NUM> 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 <NUM>% 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 <NUM>-fold increase of the translocation dwell time, <FIG>.

By counting the percentage of the triple peak events of all triple peak and double peak events, the binding ratio can be calculated, <FIG>. As expected, the proportion of triple peak signature raised with the addition of thrombin. In this experiment, we varied the concentration of thrombin from <NUM> to <NUM> while the concentration of the dimer remained <NUM>. From the statistics of normalised peak position (<FIG>), 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 <NUM> pM change of the thrombin; the triple/double peak ratio will change <NUM>%. Further, to validate the selectivity of the AuNP dimer thrombin carrier, <NUM> lysozyme was incubated with <NUM> thrombin carrier and subsequent nanopore detection of the mixture was performed, <FIG>. There is no sign of triple peak signature but the double peak events, which are corresponding to the unbounded dimer carrier.

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 (<15kDa). 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.

Herein, based on our previous work<NUM>,<NUM>, 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>) and this changing can be recorded when the molecules pass through the nanopore.

In this case, we use the AuNP monomer antigen probes to detect procalcitonin (PCT; M. <NUM> kDa; pl of <NUM>) 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 <NUM>. Therefore, with an antibody-antigen-antibody sandwich linker which is approximately <NUM>, the substructure of the dumbbell molecule (the centre of one AuNP to the other is around <NUM>) can be distinguished by nanopores with sub-<NUM> effective length.

A comparison between the translocation of <NUM> AuNP monomers (<NUM>% are functionalized by PCT mAb, and <NUM>% are functionalized by smAb) and the assembled dumbbell complex after adding <NUM> PCT, was shown in <FIG>. Without the presence of PCT, no double peak signal but a single peak signal was observed during the translocation of <NUM> AuNP monomers with <NUM> KCl, pH <NUM>. With the addition of <NUM> PCT, about <NUM>% 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 <NUM>-<NUM>). As a negative control, no double peak signatures observed when PCT was replaced by other antigens, for example, insulin, <FIG>.

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 <NUM> while the concentration of PCT was ranging from <NUM> to <NUM>. As expected, the percentage of the single peak (located on <NUM> of the normalised peak position) decreased whereas the proportion of double peak signatures (located on <NUM> and <NUM> of the normalised peak position) raised with the increasing of PCT, Fig. 4d(i). The results were further confirmed by TEM (<FIG>(ii)), which provided visualised evidence that the proportion of dimer increased with more addition of PCT.

In bacterial infections, sepsis, severe sepsis and septic shock, PCT in plasma concentrations increases from <NUM> to more than <NUM> 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 < <NUM> ng/ml, antibiotic therapy is strongly discouraged; if PCT level > <NUM> 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> inset).

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. [<NUM>] However, the signal of these short fragments (typically <NUM>-<NUM> bases) is hard to detect directly with solid-state nanopores due to the high-speed translocation and low signal-to-noise ratio, <FIG>. Here, we use AuNP dimer self-assembly to amplify this translocation signal, leading to very efficient miRNA detection at the single-molecule level.

In this study, we use AuNP molecular probes for the detection of miRNA-<NUM>. miRNA-<NUM> 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. [<NUM>] 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-<NUM> has the potential to become a useful biomarker.

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

The specificity of the molecular probes was verified by detecting miRNA-200a, which is also in the miR-<NUM> family and share seed sequences differing in only two nucleotides when compared with miRNA-<NUM>, <FIG>. The full recognition of miRNA-<NUM> 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>. The result is further confirmed by TEM, <FIG>, <FIG>. Such high selective capability probably benefits from the dimerization mechanism. For example, for miRNA-<NUM>, 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.

Although nanoparticle-based superstructures have already been reported and some of them are detected by nanopore sensing<NUM>, many of these tests are based on sensing the excluded 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.

Claim 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;
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; and
wherein the analyte is a protein.