Fluorescence enhancement using quenchers

Abstract

The present disclosure relates to a method of detecting binding of a labeled species to an analyte of interest. In some embodiments, the method comprises detecting binding of the labelled species to the analyte of interest within an area of electrical field enhancement, in the presence of a quencher.

Description

Atorney Docket No.: 67649-702601FLUORESCENCE ENHANCEMENT USING QUENCHERSCross Reference To Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 700,497 filed on September 27, 2024, the entirety of which is incorporated herein by reference.Technical Field

[0002] The present disclosure relates generally to methods of detecting binding of a labelled species to an analyte of interest within an area of electrical field enhancement, in the presence of a quencher.Background

[0003] Accurate and quantifiable detection of the binding of a labelled species to an analyte of interest is a feature of many analytical methods, particularly those used in the biological sciences, such as sensing assays and methods of sequencing nucleic acids and polypeptides. The increasing utility and efficacy of such detection and quantification methods has played an important role in the rapid development and capacity of the biological sciences. In particular, DNA sequencing technologies have revolutionized the biological sciences and continues to impact multiple industries including medicine, agriculture, and biotechnology. As a result, the DNA sequencing market is predicted to reach $60 billion by 2030. Nonetheless, serious challenges exist for the current wave of industry -leading high-throughput sequencing methods including the need for large quantities of DNA, limitations on the maximum DNA length that can be continuously read, and the computational resources needed to build the sequence from raw data. A further challenge that exists for many of the existing high-throughput sequencing methods, and which similarly impedes other analytical methods which require accurate detection of binding events between labelled species and analytes of interests in complex environments, is low signal to noise ratio. In this regard, the quality of the detected signal is often critical to accurate identification of the analyte and / or its quantification, and this may be further complicated in some methods by the need to discriminate between multiple detectable analytes. For these and other reasons, increasing the signal-to-noise ratio is a coveted goal for many analytical methods, but this is particularly the case for sensing (e.g., biosensing) and DNA / protein sequencing applications where data integrity is critical.Atorney Docket No.: 67649-702601Summary

[0004] Regions of electrical field enhancement (also referred to herein as “enhancement regions”) generated by e.g. surface structures, waveguides, nanoparticles or other metal structures, can be used to enhance the local photonic field at the point of excitation of a fluorescent molecule, and in doing so can benefit methods and assays that involve the detection of fluorescently-labelled molecules. By localising a fluorescently-labelled molecule within an enhancement region during electromagnetic irradiation, it is possible to influence the intensity and dynamic of the interaction between molecules and light, making it possible to increase the emitting rate of fluorescent molecules within the enhancement region.

[0005] Whilst enhancement regions (and other strategies for signal amplification) are being explored and / or applied in a range of analytical assays which rely on the detection of binding events between fluorescently-labelled species and analytes of interests, it remains a challenge to reduce or eliminate background noise generated from other components in the sample or assay composition which may also benefit from signal amplification. However, the inventors have surprisingly found that an increase in signal-to-noise ratio (SNR) can be achieved when a fluorescently labelled species within an enhancement region is coupled and / or in close proximity to a quencher which attenuates or reduces the light emitted by the fluorescently- labelled species. The inventors have also found that this holds true even at equivalent concentrations of fluorescent label and quencher. The discovery is counterintuitive, because quenchers are widely understood to “quench” or attenuate the fluorescence emissions from a fluorescent species when excited, and thus a reduction in the intensity of the observable fluorescence signal would be expected when the fluorophore is in proximity to a quencher. Despite this conventional wisdom, the inventors have shown experimentally, and for the first time, that the SNR associated with a binding event between a fluorescently-labelled species and an analyte of interest within in an area of electrical field enhancement is increased in the presence of a quencher, compared to the SNR associated with an equivalent binding event in the absence of a quencher. Without being bound to any one theory, the inventors believe that this observed increase in SNR may be the result of engineering the spontaneous decay rate of the fluorophore attached to the fluorescently-labelled species in two ways at the same time. Firstly, the positioning the quencher in close proximity to the fluorophore increases the non- radiative decay rate of the fluorophore, leading to a reduction in quantum yield. Secondly, positioning of the fluorophore within an enhancement region increases the rate of radiative decay, which leads to an increase in quantum yield within the enhancement region. TheAtorney Docket No.: 67649-702601 inventors postulate that combining these two effects lead to reduced background noise and a recovered signal within the enhancement region, because the increase in rate of radiative decay for fluorophores within the enhancement region is relatively greater than the increase in rate of non-radiative decay for fluorophores which are quenched. This surprising finding may provide a number advantages in the context of analyte detection assays e.g., biosensing assays and DNA / protein sequencing methods, including inter alia (i) increasing efficiency for detecting fluorescent signal associated with an analyte of interest and / or binding event, (ii) improving accuracy of detection and subsequent data integrity, (iii) reducing the excitation power that may be required in a detection assay without adversely compromising fluorescent signal detection efficiency, (iv) enabling the use of less efficient and / or sophisticated equipment (e.g., optical devices) for detecting fluorescent signal, and / or (iv) allowing for increased concentration of fluorescently-labelled species in assay compositions without a corresponding increase in background noise. More specifically, in the context of DNA / RNA sequencing methods e.g., single molecule sequencing, as well as in the context of protein sequencing methods which rely on detection of fluorescence associated with binding events and / or Edman degradation, these and other advantages may result in greater accuracy, faster sequencing and / or longer reads. Similarly, in the context of biosensing assays, such as those used in diagnostic applications, the above advantages may result in greater accuracy and sensitivity for detecting target analytes, particularly when present at lower concentrations and / or faster assay performance.

[0006] Accordingly, the present disclosure provides a method of detecting binding of a labelled species to an analyte of interest, comprising:(I) contacting one or more labelled species, each comprising a fluorophore, with the analyte of interest in the presence of one or more quenchers of the fluorophore(s) and within an area of electrical field enhancement, wherein:(i) the or each labelled species comprises one or more quenchers, and the fluorophore and one or more quenchers are positioned relative to one another on the labelled species such that the one or more quenchers attenuate the fluorescence emission intensity of the fluorophore(s) when the or each fluorophore is excited; and / orAtorney Docket No.: 67649-702601(ii) the one or more quenchers are provided separately at a concentration that attenuates the fluorescence emission intensity of the fluorophore(s) when the or each fluorophore is excited, wherein the labelled species has a distinct fluorescence emission signature which is enhanced when the fluorophore is excited within the area of electrical field enhancement; and(II) detecting the enhanced distinct fluorescence emission signature of the labelled species within the area of electrical field enhancement following excitation of the fluorophore with light at a wavelength corresponding to an excitation wavelength of the fluorophore, wherein detection of the enhanced distinct fluorescence emission signature indicates that the labelled species is bound to the analyte of interest.

[0007] In some examples, the distinct fluorescence emission signatures of the one or more labelled species are enhanced within the area of electrical field enhancement relative to the distinct fluorescence emission signatures of corresponding labelled species outside of the area of electrical field enhancement at equivalent fluorophore concentrations. For example, the distinct fluorescence emission signatures of the one or more labelled species may be enhanced by two times or more (e.g., by about three times or more, or by about four times or more, or by about five times or more, or by about six times or more, or by about seven times or more, or by about eight times or more or by about nine times or more) as compared to the distinct fluorescence emission signatures of corresponding labelled species outside of the area of electrical field enhancement. In some examples, the distinct fluorescence emission signatures of the labelled species are enhanced by an order of magnitude or more (e.g., about lOx or more, or about 20x or more, or about 30x or more, or about 40x or more, or about 50x or more, or about 60x or more, or about 70x or more, or about 80x or more, or about 90x or more, or about lOOx or more) as compared to the distinct fluorescence emission signatures of corresponding labelled species outside of the area of electrical field enhancement. In further examples, the distinct fluorescence emission signatures of the labelled species are enhanced by at least about lOOx or more (e.g., at least about 150x, or at least about 200x, or at least about 250x, or at least about 300x, or at least about 350x, or at least about 400x, or at least about 450x, or at least about 500x, or at least about 550x, or at least about 600x, or at least about 650x, or at least about 700x, or at least about 750x, or at least about 800x, or at least about 850x, or at least about 900x, or at least about 950x, or at least about lOOOx) as comparedAtorney Docket No.: 67649-702601 to the distinct fluorescence emission signatures of corresponding labelled species outside of the area of electrical field enhancement.

[0008] In some examples, the signal-to-noise ratio (SNR) of the one or more labelled species is increased within the area of electrical field enhancement compared to performance of the method using the corresponding labelled species without the one or more quenchers at equivalent fluorophore concentrations. For example, the SNR of the one or more labelled species may be increased within the area of electrical field enhancement by two times or more (e.g., by about three times or more, or by about four times or more, or by about five times or more, or by about six times or more, or by about seven times or more, or by about eight times or more or by about nine times or more) as compared to the SNR when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations. In some examples, the SNR of the one or more labelled species is increased within the area of electrical field enhancement by an order of magnitude or more (e.g., about lOx or more, or about 20x or more, or about 30x or more, or about 40x or more, or about 50x or more, or about 60x or more, or about 70x or more, or about 80x or more, or about 90x or more, or about lOOx or more) as compared to the SNR when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations.

[0009] In accordance with any one of the foregoing examples, the background noise of the one or more labelled species outside the area of electrical field enhancement may be reduced as compared to the background noise of the one or more labelled species outside the area of electrical field enhancement when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations. For example, the background noise of the one or more labelled species outside the area of electrical field enhancement may be reduced by two times or more (e.g., by about three times or more, or by about four times or more, or by about five times or more, or by about six times or more, or by about seven times or more, or by about eight times or more or by about nine times or more) as compared to the background noise when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations. In some examples, the background noise is reduced by an order of magnitude or more as compared to the background noise when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations.Atorney Docket No.: 67649-702601

[0010] In one example, at least one or each fluorophore attached to the one or more labelled species is present at a concentration of at least about lOnM. For example, at least one fluorophore attached to the one or more labelled species may be present at a concentration of between about lOnM to about lOOnM (e.g, such as about 20nM, or about 30nM, or about 40nM, or about 50nM, or about 60nM, or about 70nM, or about 80nM, or about 90nM, or about lOOnM).

[0011] In some examples, at least one or each fluorophore attached to the one or more labelled species has a fluorescence lifetime of at least 0.1ns. For example, the or each fluorophore attached to the one or more labelled species may have a fluorescence lifetime of between about 0.1ns and about 500ns. In one example, the or each fluorophore attached to the one or more labelled species may have a fluorescence lifetime of between about 0.1ns and about 5ns. In one example, the or each fluorophore attached to the one or more labelled species may have a fluorescence lifetime of between about Ins and about 10ns (e.g., such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ns). In another example, the or each fluorophore attached to the one or more labelled species has a fluorescence lifetime of between about 10ns and about 100ns (e.g., between about 10ns to about 30ns, or between about 10ns to about 50ns, or between about 10ns to about 70ns). According to some examples, the enhanced distinct fluorescence emission signature of the labelled species may be distinct due to the fluorescence lifetime of the fluorophore attached to the labelled species.

[0012] In one example, each fluorophore has a peak emission wavelength independently selected from an emission wavelength in the visible spectrum, an emission wavelength in the ultraviolet (UV) spectrum, an emission wavelength in the infrared (IR) spectrum, and an emission wavelength in the near-IR spectrum. In one example, each fluorophore has a peak emission wavelength independently selected from an emission wavelength between about 350nm and 850nm (e.g, between about 350nm and about 650nm). In some examples, at least one or each fluorophore attached to the one or more labelled species has a Stokes shift of greater than or equal to about 5 nm. For example, at least one or each fluorophore attached to the one or more labelled species has a Stokes shift of between about 5nm to about 30nm (e.g., such 6 nm, or 7 nm, or 8 nm, or about 9 nm, or about 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, or 29 nm or 30nm).

[0013] In some examples, at least one or each fluorophore attached to the one or more labelled species has a quantum yield of between about 1% and about 50%. For example, at least oneAtorney Docket No.: 67649-702601 or each fluorophore attached to the one or more labelled species may have a quantum yield of between about 1% and about 20%. For example, at least one or each fluorophore attached to the one or more labelled species may have a quantum yield of between about 10% and about 30%.

[0014] In other examples, at least one or each fluorophore attached to the one or more labelled species has a quantum yield of greater than about 50% (e.g., greater than or equal to about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0015] In some examples, at least one or each fluorophore attached to the one or more labelled species is an organic fluorophore.

[0016] In some examples, the or each fluorophore is selected independently from the group consisting of: fluorescein, rhodamine, cyanine, di pyrrom ethene, naphthalene, xanthene, anthracene, squaraine and triangulenium.

[0017] In one example, the or each fluorophore is independently selected from the group consisting of: ATTO 565, ATTO 655, Acridine Orange, Acridine Yellow, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, BODIPY 500 / 510, BODIPY 530 / 550, BODIPY FL, BODIPY TR-X, Cascade Blue, Coumarin 6, CY2, CY3B, CY3, CY3.5, CY5, CY5.5, Dansyl, DAPI, DPH, Erythrosin, Ethidium Bromide, Fluorescein, FITC, FURA-2, GFP, Hoechst 33258, Hoechst 33342, HPTS, Indocyanine Green, KU530, KU560, Laurdan, Lucifer Yellow, Nile Red, Oregon Green, Prodan, Pyrene, Rhodamine 101, Rhodamine 110, Rhodamine 123, Rhodamine 6G, Rhodamine B, Ru(bpy)3[PFe]2, Ru(bpy)2(dcpby)[PFe]2, SeTau dyes such as SeTau-380, SeTau-404, SeTau-405, SeTau-647, SeTau-425, SITS, SNART, Stilbene SITS, SITA, Texas Red, TOTO-1, YOYO-1, and YOYO-3.

[0018] In one example, the one or more quenchers are independently selected from the group consisting of: BHQ-0, BHQ-1, BHQ-2, BHQ-3, BHQ-10, QSY7, QSY9, QSY21, QSY35, Dabeyl, Dabsyl, Cy5Q, Cy7Q, DYQ-660, DYQ-661, 540Q, 580Q, and 612Q.

[0019] The area of electrical field enhancement may be produced by any means known in the art. In some examples, the area of electrical field enhancement is produced by nanoparticles (such as, for example, a single nanoparticle or multiple nanoparticles, including multiple nanoparticle structures), surface structures (such as nanostructured surfaces), nanophotonicAtorney Docket No.: 67649-702601 confinement structure (such as zero-mode waveguides (ZMW) or and microcavities) and combinations thereof.

[0020] In one example, the area of electrical field enhancement is produced by one or more nanostructured surfaces.

[0021] In one example, the area of electrical field enhancement is produced by one or more nanophotonic confinement structures. For example, the nanophotonic confinement structure may be a ZMW.

[0022] In one example, the area of electrical field enhancement is produced by one or more plasmonic nanoparticles. For example, the area of electrical field enhancement may be produced by a plasmonic hotspot defined between two plasmonic nanoparticles. In some examples, the plasmonic hotspot may be produced by a plasmonic nanoantenna. For example, the plasmonic hotspot may be defined between two plasmonic nanoparticles located on the plasmonic nanoantenna, and the plasmonic hotspot is defined between two plasmonic nanoparticles located on the plasmonic nanoantenna. For example, a plasmonic hotspot may be provided by a plasmonic nanoantenna comprising: two plasmonic nanoparticles; and a nanoscopic nucleic acid scaffold; wherein:(a) the two plasmonic nanoparticles are bound to the nanoscopic nucleic acid scaffold and positioned relative to one another such that a plasmonic hotspot exists between the two plasmonic nanoparticles; and(b) the plasmonic hotspot comprises a region unoccupied by the nanoscopic nucleic acid scaffold in which binding of the one or more labelled species to the analyte of interest occurs.

[0023] However, in other examples the plasmonic nanoantenna comprises more than two plasmonic nanoparticles, each bound to the nanoscopic nucleic acid scaffold and positioned relative to one another such that a plasmonic hotspot exists between the plasmonic nanoparticles. For example, the plasmonic nanoantenna may comprise three plasmonic nanoparticles. For example, the plasmonic nanoantenna may comprise four plasmonic nanoparticles. For example, the plasmonic nanoantenna may comprise five plasmonic nanoparticles. For example, the plasmonic nanoantenna may comprise six plasmonic nanoparticles. For example, the plasmonic nanoantenna may comprise seven plasmonic nanoparticles. For example, the plasmonic nanoantenna may comprise eight plasmonicAtorney Docket No.: 67649-702601 nanoparticles. For example, the plasmonic nanoantenna may comprise nine plasmonic nanoparticles. For example, the plasmonic nanoantenna may comprise ten or more plasmonic nanoparticles.

[0024] In some examples, the region of the plasmonic hotspot unoccupied by the nanoscopic nucleic acid scaffold may be defined by a three-dimensional space having a length (L) between the two plasmonic nanoparticles, a height (H) at a midpoint (L / 2) that is perpendicular to (L), and a width (W) at L / 2 that is perpendicular to L and H wherein at L / 2, W is selected from 20 nm to about 100 nm and H is selected from 10 nm to about 100 nm wherein L is measured at the shortest distance between the two plasmonic nanoparticles. In some examples, L is selected from 20 nm to 100 nm. In other examples, L is selected from 20 nm to 50 nm.

[0025] In other examples, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold may be defined in terms of volume, for example, as having a volume of at least about 1 zL, such as from about IzL to about 10 zL, or from about 5zL to about 10 zL. In certain examples, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold has a volume which is greater than or equal to about 10 zL. For example, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold has a volume of about 10 zL to about 50 zL. For example, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold may have a volume of about 20 zL to about 40 zL.

[0026] In some examples, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold is less than or equal to 50% of the plasmonic hotspot. However, in other examples, the region unoccupied by the nanoscopic nucleic acid scaffold is more than 50% of the plasmonic hotspot. In some examples, the region unoccupied by the nanoscopic nucleic acid scaffold is 60% or more of the plasmonic hotspot. In certain examples, the plasmonic hotspot is substantially unoccupied by the nanoscopic nucleic acid scaffold. For example, the region unoccupied by the nanoscopic nucleic acid scaffold may be 70% or more of the plasmonic hotspot. In some examples, the region unoccupied by the nanoscopic nucleic acid scaffold is 75% or more of the plasmonic hotspot. In some examples, the region unoccupied by the nanoscopic nucleic acid scaffold is 80% or more of the plasmonic hotspot. In some examples, the region unoccupied by the nanoscopic nucleic acid scaffold is 85% or more of the plasmonic hotspot. In some examples, the region unoccupied by the nanoscopic nucleic acid scaffold is 90% or more of the plasmonic hotspot.Atorney Docket No.: 67649-702601

[0027] In each of the foregoing examples describing plasmonic nanoantenna for use in the method of the disclosure, the region unoccupied by the nanoscopic nucleic acid scaffold may take different shapes or form. In one example, the region unoccupied by the nanoscopic nucleic acid scaffold may be amorphous. Alternatively, the region unoccupied by the nanoscopic nucleic acid scaffold may have a substantially defined shape. For example, the region unoccupied by the nanoscopic nucleic acid scaffold may be amorphous, spherical or cubic.

[0028] The average interparticle distance between the plasmonic nanoparticles positioned on the nanoscopic nucleic acid scaffold may also be varied as required to optimise the size and / or strength of the area of electrical field enhancement. In one example, the average interparticle distance between the plasmonic nanoparticles is about 10 nm to about 100 nm, wherein the interparticle distance between two particles is determined as the shortest distance between the surfaces of the respective particles. For example, the average interparticle distance between the plasmonic nanoparticles may be about 10 nm to 80 nm. For example, the average interparticle distance between the plasmonic nanoparticles may be about 10 nm to 50 nm. For example, the average interparticle distance between the plasmonic nanoparticles may be about 20 nm to 80 nm. For example, the average interparticle distance between the plasmonic nanoparticles may be about 20 nm to 50 nm. For example, the average interparticle distance between the plasmonic nanoparticles may be about 30 nm to 50 nm. For example, the average interparticle distance between the plasmonic nanoparticles may be about 20 nm to 40 nm. For example, the average interparticle distance between the plasmonic nanoparticles may be about 30 nm to 40 nm. In one particular example, the plasmonic nanoparticles have an average interparticle distance of about 30 nm.

[0029] The plasmonic nanoparticles of the plasmonic nanoantenna described herein may be formed of any material which possesses plasmonic resonance properties. Suitable plasmonic nanoparticles include, but are not limited to, metallic nanoparticles, metal alloy nanoparticles, polymeric nanoparticles and derivatives and composites thereof. In one example, each of the plasmonic nanoparticles are independently selected from metallic nanoparticles. For example, each of the two or more plasmonic nanoparticle is a metallic nanoparticle independently selected from the group consisting of gold nanoparticles, silver nanoparticles, aluminium nanoparticles, copper nanoparticles, bismuth nanoparticles, nickel nanoparticles, palladium nanoparticles and platinum nanoparticles or a nanoparticle formed of an alloy of any one thereof. In some examples, the metallic nanoparticles are solid and formed whollyAtorney Docket No.: 67649-702601 of the metallic substance. In other examples, the metallic nanoparticles are formed wholly of the metallic substance but have a hollow core. In other examples, the metallic nanoparticles comprise a core (e.g., a glass, polymer or composite core) which is coated in the metallic substance to form a metallic surface. In one example, the plasmonic nanoparticles are gold nanoparticles or gold alloy nanoparticles. In one example, the plasmonic nanoparticles are silver nanoparticles or silver alloy nanoparticles. In other examples, the plasmonic nanoparticles are formed of a non-metallic material or metal oxide which has been doped to increase the total carrier load of the nanoparticle and thereby increase its plasmon resonance.

[0030] The plasmonic nanoparticles of the plasmonic nanoantenna described herein may also vary in their shape and geometry. Accordingly, the plasmonic nanoparticles may be selected independently from nanospheres, nanorods, nanoprisms, nanocubes, nanoshells, nanotubes, or nanostars. The plasmonic nanoparticles may be the same shape or they may be heterogeneous. In one example, the plasmonic nanoparticles are nanospheres.

[0031] The plasmonic nanoparticles of the plasmonic nanoantenna described herein may vary in their dimensions. In one example, each of the plasmonic nanoparticles has a largest diameter of about 5 nm to about 500 nm. In one example, each of the plasmonic nanoparticles has a largest diameter of about 10 nm to about 250 nm. In one example, each of the plasmonic nanoparticles has a largest diameter of about 20 nm to about 200 nm. In one example, each of the plasmonic nanoparticles has a largest diameter of about 50 nm to about 150 nm. In one example, each of the plasmonic nanoparticles has a largest diameter of about 80 nm to about 120 nm. In one particular example, each of the plasmonic nanoparticles has a largest diameter of about 80 nm. In another particular example, each of the plasmonic nanoparticles has a largest diameter of about 100 nm. In yet another particular example, each of the plasmonic nanoparticles has a largest diameter of about 120 nm.

[0032] In some examples, the plasmonic nanoparticles have the same largest diameter. Whereas in other examples, the largest diameter differs between plasmonic nanoparticles on the same plasmonic nanoantenna. In accordance with an example in which the largest diameter differs between plasmonic nanoparticles on the same plasmonic nanoantenna, the plasmonic nanoparticles may have an average largest diameter of about 5 nm to about 500 nm. For example, the plasmonic nanoparticles on the plasmonic nanoantenna may have an average largest diameter of about 10 nm to about 250 nm. For example, the plasmonic nanoparticles on the plasmonic nanoantenna may have an average largest diameter of about 20 nm to about 200 nm. For example, the plasmonic nanoparticles on the plasmonicAtorney Docket No.: 67649-702601 nanoantenna may have an average largest diameter of about 50 nm to about 150 nm. For example, the plasmonic nanoparticles on the plasmonic nanoantenna may have an average largest diameter of about 80 nm to about 120 nm. In one particular example, the plasmonic nanoparticles on the plasmonic nanoantenna may have an average largest diameter of about 80 nm. In one particular example, the plasmonic nanoparticles on the plasmonic nanoantenna may have an average largest diameter of about 100 nm. In one particular example, the plasmonic nanoparticles on the plasmonic nanoantenna may have an average largest diameter of about 120 nm.

[0033] In one particular example, the plasmonic nanoantenna described herein comprises gold or gold-coated nanoparticles (e.g., a nanosphere) having an average largest diameter of about 100 nm.

[0034] In each of the foregoing examples describing the use of plasmonic nanoantenna in the method of the disclosure, the plasmonic nanoparticles may be coated with a coating. Preferably, the coating substantially covers the surface of each of the plasmonic nanoparticles. In one example, the coating covers about 75% or more of the surface of each of the plasmonic nanoparticles. In one example, the coating covers about 80% or more of the surface of each of the plasmonic nanoparticles. In one example, the coating covers about 85% or more of the surface of each of the plasmonic nanoparticles. In one example, the coating covers about 90% or more of the surface of each of the plasmonic nanoparticles. In one example, the coating covers about 95% or more of the surface of each of the plasmonic nanoparticles.

[0035] The coating may reduce aggregation of the plasmonic nanoparticles relative to plasmonic nanoparticles without the coating. In one example, the coating reduces aggregation of the plasmonic nanoparticles by 35% or more relative to plasmonic nanoparticles without the coating. In one example, the coating reduces aggregation of the plasmonic nanoparticles by 50% or more relative to plasmonic nanoparticles without the coating. In one example, the coating reduces aggregation of the plasmonic nanoparticles by 65% or more relative to plasmonic nanoparticles without the coating.

[0036] In one example, the coating of the plasmonic nanoparticles comprises a plurality of oligonucleotides. In some examples, each nucleotide in each oligonucleotide of the plurality of oligonucleotides is selected from pyrimidine nucleotides. In one example, each oligonucleotide of the plurality of oligonucleotides comprises about 20 nucleotides to about 50 nucleotides. In one example, each oligonucleotide of the plurality of oligonucleotides comprises about 20 nucleotides to about 30 nucleotides.Atorney Docket No.: 67649-702601

[0037] In certain examples, the nanoscopic nucleic acid scaffold of the plasmonic nanoantenna is be composed substantially of double stranded DNA. For example, the double stranded DNA may be parallel interconnected strands of double helices. In some examples, the nanoscopic nucleic acid scaffold is composed of DNA origami structures e.g., 3D DNA origami structures. In one example, the DNA origami structure comprises a M13mpl8- derived scaffold strand and complementary staple strands. In certain example examples, at least a portion of the DNA origami structures are assembled to form a U-shaped or curved structure.

[0038] In one example, the nanoscopic nucleic acid scaffold of the plasmonic nanoantenna comprises a first face to which one of two plasmonic nanoparticles is anchored and a second face to which the second of two plasmonic nanoparticles is anchored. The first face of the scaffold and the second face of the scaffold may be on the same or adjacent sides of the nanoscopic nucleic acid scaffold. Alternatively, the first face of the scaffold and the second face of the scaffold may be on opposite sides of the nanoscopic nucleic acid scaffold.

[0039] In accordance with examples in which the plasmonic nanoantenna used in the method of the disclosure comprises two plasmonic nanoparticles, one plasmonic nanoparticle is anchored to the first face of the scaffold through one or more nucleic acid linkers and the other plasmonic nanoparticle is anchored to the second face of the scaffold through one or more nucleic acid linkers, wherein each nucleic acid linker is formed by an oligonucleotide coated on the surface of one of the plasmonic nanoparticles which is hybridised to a polynucleotide forming part of, or extending from, the nanoscopic nucleic acid scaffold, and wherein the sequences of the respective oligonucleotides and polynucleotides which are capable of hybridising are complementary or substantially complementary to one another. In accordance with this example, the nanoscopic nucleic acid scaffold comprises one or more polynucleotides extending from the first face of the scaffold, each comprising a sequence which is complementary to a sequence of one or more oligonucleotides coating one of the two plasmonic nanoparticles. Additionally, the nanoscopic nucleic acid scaffold comprises one or more polynucleotides extending from the second face of the scaffold, each comprising a sequence which is complementary to a sequence of one or more oligonucleotides coating one of the two plasmonic nanoparticles. The polynucleotides forming part of, or extending from, the nanoscopic nucleic acid scaffold, may each be a single-stranded DNA. Preferably, the polynucleotides extending from the first and second faces of the scaffold each comprise aAtorney Docket No.: 67649-702601 region of sufficient length and complementarity to hybridise to the oligonucleotides coating the surface of the plasmonic nanoparticles. In one example, the polynucleotides extending from the first and second faces of the scaffold each comprise about 20 nucleotides to about 50 nucleotides. In another example, the polynucleotides extending from the first and second faces of the scaffold comprises about 20 nucleotides to about 30 nucleotides. In each of these examples, the polynucleotides extending from the first and second faces of the scaffold each comprise a region of about 10 to about 25 nucleotides in length which is capable of hybridising to a region of corresponding length within the oligonucleotides coating the surface of the plasmonic nanoparticles.

[0040] In accordance with examples in which the area of electrical field enhancement is produced by a plasmonic nanoantenna as described herein, the method may comprises using an array of plasmonic nanoantenna described herein. Each plasmonic nanoantenna in the array may be configured to detect an analyte of interest. The analytes detected by the plasmonic nanoantenna of the array may be the same or different.

[0041] In another example, the area of electrical field enhancement is produced by a nanostructured surface. In some examples, the nanostructured surface may be configured for detecting a single analyte of interest or multiple analytes of interest. For example, where the nanostructured surface is configured for detecting multiple analytes of interest, the nanostructured surface may be configured as an array comprising multiple nanostructures.

[0042] In yet another example, the area of electrical field enhancement is produced by a zeromode waveguide. In some examples, the method may comprises using an array of zero-mode waveguides. Each zero-mode waveguide in the array may be configured to detect a single analyte of interest. The analytes detected by the zero-mode waveguides of the array may be the same or different.

[0043] In each of the foregoing examples, the or each labelled species has distinct fluorescence emission signature which is enhanced when the fluorophore is excited within the area of electrical field enhancement. Distinct fluorescence emission signatures of the labelled species may be distinguishable (e.g., from other emission signatures in the method, such as those of other labelled species) based on one or more of the following characteristics: peak emission wavelength, fluorescence emission intensity, fluorescence emission lifetime, duration of fluorescence (e.g., dwell time), duration between successive fluorescence emissions, or any combinations thereof.Atorney Docket No.: 67649-702601

[0044] In accordance with examples in which enhanced distinct fluorescence emission signatures are detected for multiple labelled species, the respective labelled species may be distinguished from one another based on differences in one or more of the following: peak emission wavelength, fluorescence emission intensity, fluorescence emission lifetime, duration of fluorescence (e.g., dwell time), duration between successive fluorescence emissions, or any combinations thereof.

[0045] Where multiple labelled species are contacted with the analyte of interest, they may be contacted with the analyte at the same time (e.g., introduced simultaneously), or they may be contacted with the analyte one at a time and in isolation (e.g., introduced sequentially with wash steps in between the respective contacting steps).

[0046] In some examples, two or more labelled species comprise the same fluorophore, and the enhanced distinct fluorescence emission signatures of the respective labelled species are distinguished based on differences in fluorescence emission intensity and / or differences in duration of fluorescence e.g., differences in dwell time of the labelled species.

[0047] In some examples, two or more labelled species comprise different fluorophores, and the enhanced distinct fluorescence emission signatures of the respective labelled species are distinguished based on differences in peak emission wavelength, fluorescence emission intensity, fluorescence emission lifetime, or any combinations thereof of the labelled species.

[0048] For example, where the method comprises detecting enhanced distinct fluorescence emission signatures for multiple labelled species, at least one or each of the labelled species is attached to a fluorophore having a distinct peak emission wavelength. For example, the distinct peak emission wavelengths of the multiple labelled species may be separated by 10 nm or more relative to each other.

[0049] In each of the foregoing examples, detecting the enhanced distinct fluorescence emission signatures at (II) may be performed using fluorescence microscopy or a fluorometer. For example, detecting the enhanced distinct fluorescence emission signatures at (II) may be performed using total internal reflection fluorescence (TIRF) microscopy.

[0050] In certain examples, the method of detecting binding of a labelled species to an analyte of interest as described herein is a method of sequencing a nucleic acid molecule. According to one example in which the method described herein is a method of sequencing a nucleic acid molecule: the or each labelled species is a labelled nucleotide;Atorney Docket No.: 67649-702601 the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a mixture of labelled nucleotides for a time and under conditions such that one of the labelled nucleotides binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, wherein the complementary nucleotide is positioned within an active site of the nucleic acid polymerase; wherein:(i) the mixture of labelled nucleotides comprises a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore and one or more quenchers are each linked directly or indirectly to the respective nucleotides, and are positioned relative to one another on the nucleotides such that the one or more quenchers attenuate emission intensity of the fluorophore when the fluorophore is excited,(iii) each of the labelled nucleotides A, G, T and C has a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited,(iv) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(v) the distinct fluorescence emission signatures of the labelled nucleotides are enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelled nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature; wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the labelled nucleotides sequentially bind to, and form ternaryAtorney Docket No.: 67649-702601 complexes with, the complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand.

[0051] According to another example in which the method described herein is a method of sequencing a nucleic acid molecule: the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a mixture of labelled nucleotides in the presence of one or more quenchers for a time and under conditions such that one of the labelled nucleotides binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, wherein the complementary nucleotide is positioned within an active site of the nucleic acid polymerase; wherein:(i) the mixture of labelled nucleotides comprises a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore is linked directly or indirectly to each of the nucleotides, wherein each fluorophore has an emission spectrum when excited which overlaps with an absorption spectrum of one or more of the quenchers,(iii) the one or more quenchers are not attached to the labelled nucleotides and are provided at a concentration that attenuates fluorescence emission intensity of the fluorophores when the fluorophores are excited,(iv) each of the labelled nucleotides A, G, T and C has a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited,(iv) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(v) the distinct fluorescence emission signatures of the labelled nucleotides are enhanced upon binding to, and forming a ternary complex with, aAtorney Docket No.: 67649-702601 complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelled nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature; wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the labelled nucleotides sequentially bind to, and form ternary complexes with, the complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand.

[0052] The method of sequencing a nucleic acid molecule as described herein may be a sequencing-by-synthesis (SBS, also referred to as ‘sequencing by incorporation’) method or a sequencing-by-binding (SBB) method.

[0053] In one example, the method of sequencing a nucleic acid molecule as described herein is a method of SBS, wherein: each labelled nucleotide that binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, is subsequently incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex is detected during incorporation of the labelled nucleotide into the nascent polynucleotide strand; and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures as the labelled nucleotides are bound to complementary nucleotides within the active site of the nucleic acid polymerase and sequentially incorporated into the nascent polynucleotide strand.

[0054] Where the method of sequencing a nucleic acid molecule is a method of SBS, each cycle comprising steps (I)-(II) may be repeated ‘n’ times, wherein ‘n’ is in the range of 2 to 1,000 or more. In this regard, the skilled person will appreciate that the number of cycles may be determined, in part, by the read length of the nucleic acid analyte. In one example, ‘n’ is less than 50. In one example, ‘n’ is in the range of 50-1000 (e.g., 50-100, or 50-150, or 50-Atorney Docket No.: 67649-702601200, or 50-250, or 100-300, or 100-400, or 250-500, or 250-750, or 500-1000 or more). In one example, ‘n’ is greater than 1000. In some examples, the read length of the nucleic acid analyte is equivalent to ‘n’.

[0055] In another example, the method of sequencing a nucleic acid molecule as described herein is a method of SBB, wherein: the nucleic acid analyte is primed with an oligonucleotide comprising a nucleotide at its 3’ end having a reversible blocking moiety to prevent labelled nucleotides being incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; and wherein following steps (I) and (II) in each cycle, the method further comprises the steps of:(III) performing one or more wash steps to remove labelled nucleotides;(IV) performing one or more step to remove the reversible blocker on the nucleotide at the 3’ end of the nascent polynucleotide strand, such that the nucleotide at the 3’ end of the nascent polynucleotide strand comprises a 3’ hydroxyl group which is capable of forming a phosphodiester bond with an adjacent nucleotide during incorporation by nucleic acid polymerase;(V) contacting the nucleic acid polymerase positioned within the area of electrical field enhancement with an unlabeled nucleotide comprising a reversible blocking moiety, wherein the unlabeled nucleotide comprises the same nucleotide species as the labelled nucleotide identified at step (II) such that the unlabeled nucleotide is incorporated by the nucleic acid polymerase into the nascent polynucleotide strand at its 3’ terminus;(VI) performing a wash step to remove all remaining unlabeled nucleotide, and(VII) repeating steps (I)-(VI) ‘n’ times to determine the order of enhanced distinct fluorescence emission signatures from labelled nucleotides as they sequentially bind to, and form ternary complexes with, complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand, wherein the sequence of the nucleic acid analyte is determined based on the order of enhanced distinct fluorescence emission signatures detected during the polymerase-based synthesis of the nascent polynucleotide strand.

[0056] Where the method of sequencing a nucleic acid molecule is a method of SBB, each cycle comprising steps (I)-(VI) may be repeated ‘n’ times, wherein ‘n’ is in the range of 2 toAtorney Docket No.: 67649-702601100,000 or more. In this regard, the skilled person will appreciate that the number of cycles may be determined, in part, by the read length of the nucleic acid analyte. In one example, ‘n’ is less than 50. In one example, ‘n’ is in the range of 50-1000 (e.g., 50-100, or 50-150, or 50-200, or 50-250, or 100-300, or 100-400, or 250-500, or 250-750, or 500-1000). In one example, ‘n’ is in the range of 500-10000 (e.g., 500-1000, or 500-1500, or 500-2000, or 500- 2500, or 1000-3000, or 1000-4000, or 2500-5000, or 2500-7500, or 5000-10000, or 7500- 10000). In one example, ‘n’ is in the range of 5000-25000 (e.g., 5000-7500, or 7500-10000, or 10000-12500, or 12500-15000, or 15000-17500, or 17500-20000, or 20000-22500, or 22500-25000). In one example, ‘n’ is in the range of 15000-50000 (e.g., 15000-20000, or 25000-30000, or 35000-40000, or 45000-50000). In one example, ‘n’ is in the range of 50000-100000 (e.g., 50000-60000, or 60000-70000, or 70000-80000, or 80000-90000 or 90000-100000). In some examples, the read length of the nucleic acid analyte is equivalent to ‘n’.

[0057] According to another example in which the method described herein is a method of sequencing a nucleic acid molecule, a single channel sequencing approach is employed in which a single species of labelled nucleotide is contacted with nucleic acid analyte and the polymerase at any one time, and different species of labelled nucleotide are contacted with nucleic acid analyte and the polymerase in a sequential manner. In accordance with such examples, the method of sequencing the nucleic acid molecule may be referred to as a single channel sequencing method.

[0058] In one example, a method of sequencing a nucleic acid molecule using a single channel sequencing approach may be one in which: the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a single species of labelled nucleotide for a time and under conditions suitable for the labelled nucleotide to bind to a nucleotide positioned within an active site of the nucleic acid polymerase if it is complementary thereto, thereby forming a ternary complex with the complementary nucleotide and the nucleic acid polymerase; wherein:Atorney Docket No.: 67649-702601(i) the single species of labelled nucleotide is selected from the group of labelled nucleotides consisting of a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore and one or more quenchers are each linked directly or indirectly to the labelled nucleotide at (i), and are positioned relative to one another on the nucleotides such that the one or more quenchers attenuate emission intensity of the fluorophore when the fluorophore is excited,(iii) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(iv) a distinct fluorescence emission signature of the labelled nucleotide is enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the presence or absence of the enhanced distinct fluorescence emission signature of the labelled nucleotide within the area of electrical field enhancement, wherein:(i) the presence of the enhanced distinct fluorescence emission signature is indicative that the labelled nucleotide has base-paired and formed a ternary complex with a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(ii) the absence of the enhanced distinct fluorescence emission signature is indicative that the labelled nucleotide has not base-paired, or not formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(III) performing one or more wash steps to remove the species of labelled nucleotide;(IV) where it is determined at (II) that the species of labelled nucleotide has not formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase, the method comprises repeating steps (I)-(III) in the presence of a different species of labelled nucleotide, wherein steps (I)-(III) are performed in repeated cycles with different species of labelled nucleotides until it is determined that one of the species of labelled nucleotides selected from the group consisting of labelledAtorney Docket No.: 67649-702601A, labelled G, labelled T, and labelled C base-pairs and forms a ternary complex with a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(V) repeating steps (I)-(IV) ‘n’ times to determine the order of enhanced distinct fluorescence emission signatures from labelled nucleotides as they sequentially bind to, and form ternary complexes with, complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand, wherein the sequence of the nucleic acid analyte is determined based on the order of enhanced distinct fluorescence emission signatures detected during the polymerase-based synthesis of the nascent polynucleotide strand.

[0059] In another example, a method of sequencing a nucleic acid molecule using a single channel sequencing approach may be one in which: the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a single species of labelled nucleotide in the presence of one or more quenchers for a time and under conditions suitable for the labelled nucleotide to bind to a nucleotide positioned within an active site of the nucleic acid polymerase if it is complementary thereto, thereby forming a ternary complex with the complementary nucleotide and the nucleic acid polymerase; wherein:(i) the single species of labelled nucleotide is selected from the group of labelled nucleotides consisting of a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore is linked directly or indirectly to the labelled nucleotide at (i), and wherein each fluorophore has an emission spectrum when excited which overlaps with an absorption spectrum of one or more of the quenchers,Atorney Docket No.: 67649-702601(iii) the one or more quenchers are not attached to the labelled nucleotide and are provided at a concentration that attenuates fluorescence emission intensity of the fluorophore when the fluorophore is excited;(iii) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(iv) a distinct fluorescence emission signature of the labelled nucleotide is enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the presence or absence of the enhanced distinct fluorescence emission signature of the labelled nucleotide within the area of electrical field enhancement, wherein:(i) the presence of the enhanced distinct fluorescence emission signature is indicative that the labelled nucleotide has base-paired and formed a ternary complex with a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(ii) the absence of the enhanced distinct fluorescence emission signature is indicative that the labelled nucleotide has not base-paired, or not formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(III) performing one or more wash steps to remove the species of labelled nucleotide;(IV) where if it is determined at (II) that the species of labelled nucleotide has not formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase, the method comprises repeating steps (I)-(III) in the presence of a different species of labelled nucleotide, wherein steps (I)-(III) are performed in repeated cycles with different species of labelled nucleotides until it is determined that one of the species of labelled nucleotides selected from the group consisting of labelled A, labelled G, labelled T, and labelled C base-pairs and forms a ternary complex with a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(V) repeating steps (I)-(IV) ‘n’ times to determine the order of enhanced distinct fluorescence emission signatures from labelled nucleotides as they sequentially bind to, and form ternary complexes with, complementary nucleotides within the templateAtorney Docket No.: 67649-702601 polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand, wherein the sequence of the nucleic acid analyte is determined based on the order of enhanced distinct fluorescence emission signatures detected during the polymerase-based synthesis of the nascent polynucleotide strand.|0060] In each of the foregoing examples describing sequential addition of single nucleotide species, each of the labelled nucleotides A, G, T and C may have a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited. Alternatively, the labelled nucleotides A, G, T and C may have the same fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited and differences between the nucleotide species are determined based on the knowledge of which nucleotide is being introduced in any one step.

[0061] In some examples, the order in which the different species of labelled nucleotides are contacted with the nucleic acid polymerase and the nucleic acid analyte is predetermined.

[0062] The single channel sequencing method as described herein may be a SB S method or a SBB method.

[0063] In one example, the single channel sequencing method as described herein is a SBS method, wherein: each labelled nucleotide that binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, is subsequently incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex is detected during incorporation of the labelled nucleotide into the nascent polynucleotide strand; and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures as the labelled nucleotides are bound to complementary nucleotides within the active site of the nucleic acid polymerase and sequentially incorporated into the nascent polynucleotide strand.

[0064] Where the method of single channel sequencing is a method of SBS, each cycle comprising steps (I)-(IV) may be repeated ‘n’ times, wherein ‘n’ is in the range of 2 to 1,000 or more. In this regard, the skilled person will appreciate that the number of cycles may be determined, in part, by the read length of the nucleic acid analyte. In one example, ‘n’ is lessAtorney Docket No.: 67649-702601 than 50. In one example, ‘n’ is in the range of 50-1000 (e.g., 50-100, or 50-150, or 50-200, or 50-250, or 100-300, or 100-400, or 250-500, or 250-750, or 500-1000 or more). In one example, ‘n’ is greater than 1000. In some examples, the read length of the nucleic acid analyte is equivalent to ‘n’.

[0065] In another example, the single channel sequencing method as described herein is a SBB method, wherein: the nucleic acid analyte is primed with an oligonucleotide comprising a nucleotide at its 3’ end having a reversible blocking moiety to prevent labelled nucleotides being incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; and if it is determined at (II) that the species of labelled nucleotide has formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase, the method comprises the following further steps between (III) and (IV) of any one cycle:(i) performing one or more step to remove the reversible blocker on the nucleotide at the 3’ end of the nascent polynucleotide strand, such that the nucleotide at the 3’ end of the nascent polynucleotide strand comprises a 3’ hydroxyl group which is capable of forming a phosphodiester bond with an adjacent nucleotide during incorporation by nucleic acid polymerase;(ii) contacting the nucleic acid polymerase positioned within the area of electrical field enhancement with an unlabeled nucleotide comprising a reversible blocking moiety, wherein the unlabeled nucleotide comprises the same nucleotide species as the labelled nucleotide identified at step (II) such that the unlabeled nucleotide is incorporated by the nucleic acid polymerase into the nascent polynucleotide strand at its 3’ terminus; and(iii) performing a wash step to remove all remaining unlabeled nucleotide.

[0066] Where the single channel sequencing method is a method of SBB, each cycle comprising steps (I)-(VI) may be repeated ‘n’ times, wherein ‘n’ is in the range of 2 to 100,000 or more. In this regard, the skilled person will appreciate that the number of cycles may be determined, in part, by the read length of the nucleic acid analyte. In one example, ‘n’ is less than 50. In one example, ‘n’ is in the range of 50-1000 (e.g., 50-100, or 50-150, or 50-200, or 50-250, or 100-300, or 100-400, or 250-500, or 250-750, or 500-1000). In one example, ‘n’ is in the range of 500-10000 (e.g., 500-1000, or 500-1500, or 500-2000, or 500-2500, or 1000-3000, or 1000-4000, or 2500-5000, or 2500-7500, or 5000-10000, or 7500-10000). InAtorney Docket No.: 67649-702601 one example, ‘n’ is in the range of 5000-25000 (e.g., 5000-7500, or 7500-10000, or 10000- 12500, or 12500-15000, or 15000-17500, or 17500-20000, or 20000-22500, or 22500-25000). In one example, ‘n’ is in the range of 15000-50000 (e.g., 15000-20000, or 25000-30000, or 35000-40000, or 45000-50000). In one example, ‘n’ is in the range of 50000-100000 (e.g., 50000-60000, or 60000-70000, or 70000-80000, or 80000-90000 or 90000- 100000). In some examples, the read length of the nucleic acid analyte is equivalent to ‘n’.

[0067] In each of the foregoing examples describing methods of sequencing nucleic molecules, the distinct fluorescence emission signatures of the respective labelled nucleotides may be distinguished from fluorescence emission signatures of other labelled nucleotides based on one or more of the following characteristics: peak emission wavelength, fluorescence emission intensity, fluorescence emission lifetime, duration between successive fluorescence emissions, or any combinations thereof.

[0068] In one example, each of the labelled nucleotides is attached to a different fluorophore having a distinct peak emission wavelength.

[0069] In accordance with examples in which the method of the disclosure is a method of sequencing a nucleic acid molecule, the area of electrical field enhancement may be produced by any means in the art suitable for nucleic acid sequencing, examples of which are described herein. For example, the area of electrical field enhancement may be produced by nanoparticles (such as, for example, a single nanoparticle or multiple nanoparticles, including multiple nanoparticle structures), surface structures (such as nanostructured surfaces), nanophotonic confinement structure (such as zero-mode waveguides or and microcavities) and combinations thereof.

[0070] In one example, the area of electrical field enhancement is produced by a nanophotonic confinement structure. For example, the nanophotonic confinement structure may be a zeromode waveguide (ZMW). In accordance with this example, the nucleic acid polymerase may be immobilised within the ZMW or the nucleic acid analyte to be sequenced may be immobilised within the ZMW, to ensure that the nucleic acid polymerase is positioned within the area of electrical field enhancement during synthesis of the nascent polynucleotide strand.

[0071] In another example, the area of electrical field enhancement is produced by one or more plasmonic nanoparticles. Exemplary means of producing areas of electrical field enhancement using plasmonic nanoparticles, including the production of plasmonic hotspots using plasmonic nanoparticles positioned on plasmonic nanoantennas, are describedAtorney Docket No.: 67649-702601 hereinabove and shall be taken to apply mutatis mutandis to each and every example describing the method of sequencing a nucleic acid unless stated otherwise.

[0072] In certain examples, the area of electrical field enhancement is produced by a plasmonic nanoantenna as described herein, and the nucleic acid polymerase or the nucleic acid analyte is bound to or immobilized on the nanoscopic nucleic acid scaffold of the plasmonic nanoantenna and is positioned within the region unoccupied by the nanoscopic nucleic acid scaffold. In one example, the nucleic acid polymerase is bound or immobilized on the nanoscopic nucleic acid scaffold of the plasmonic nanoantenna to ensure that the nucleic acid polymerase is positioned within the area of electrical field enhancement during synthesis of the nascent polynucleotide strand.

[0073] In one example, the nucleic acid analyte is bound to or immobilized on the nanoscopic nucleic acid scaffold to ensure that the nucleic acid polymerase is positioned within the area of electrical field enhancement during synthesis of the nascent polynucleotide strand. In some examples, the nucleic acid analyte is bound non-covalently to the nanoscopic nucleic acid scaffold, preferably through hydrogen bonding.

[0074] In accordance with examples in which a plasmonic nanoantenna described herein is used for nucleic acid sequencing, the region of the plasmonic hotspot unoccupied by the nanoscopic nucleic acid scaffold will be sufficiently large to accommodate the nucleic acid polymerase and to retain biological activity of the nucleic acid polymerase.

[0075] In some examples, the nucleic acid polymerase is bound to, or immobilized on, the nanoscopic nucleic acid scaffold through a nucleic acid linker. In some embodiments, the nucleic acid linker is double-stranded and comprises: (i) a polynucleotide which is covalently or non-covalently bound to the nucleic acid polymerase via an amino acid within the nucleic acid polymerase, and (ii) a polynucleotide forming part of, or extending from, the nanoscopic nucleic acid scaffold, wherein the polynucleotides at (i) and (ii) comprise single-stranded DNA sequences which are complementary or substantially complementary and are capable of hybridizing to one another. In one example, the polynucleotide at (i) comprises a peptide tag which is bound to the nucleic acid polymerase via an isopeptide bond formed between an amino acid within the nucleic acid polymerase and an amino acid within the peptide tag. In one example, the nucleic acid linker is bound to the nucleic acid polymerase via an amino acid within the N-terminal domain of the nucleic acid polymerase.

[0076] Each of the polynucleotides of the nucleic acid linker which binds or immobilises the nucleic acid polymerase on the nanoscopic nucleic acid scaffold comprises a region ofAtorney Docket No.: 67649-702601 sufficient length and complementarity to hybridise to one another. In one example, each polynucleotide of the nucleic acid linker comprises about 20 nucleotides to about 50 nucleotides. In another example, each polynucleotide of the nucleic acid linker comprises about 20 nucleotides to about 30 nucleotides. In each of these examples, the polynucleotides of the nucleic acid linker each comprise a region of about 10 to about 25 nucleotides in length which are complementarity and capable of hybridizing to one another.

[0077] In accordance with an example in which the nucleic acid polymerase is bound to, or immobilized on, on the nanoscopic nucleic acid scaffold of the plasmonic nanoantenna through a nucleic acid linker, the nucleic acid polymerase may be modified to express an amino acid sequence which is capable of binding to a peptide tag conjugated to the nucleic acid linker.

[0078] According to other examples, the nucleic acid analyte to be sequenced is bound to, or immobilized on, the nanoscopic nucleic acid scaffold through a nucleic acid linker. For example, the nucleic acid analyte to be sequenced may comprise an adapter sequence at its 3’ or 5’ terminus which is complementary or substantially complementary to a polynucleotide forming part of, or extending from, the nanoscopic nucleic acid scaffold, such that the adapter sequence and polynucleotide forming part of, or extending from, the nanoscopic nucleic acid scaffold are capable of hybridizing to one another. In accordance with this example, the nucleic acid polymerase may be free in solution i.e., not immobilized.

[0079] In each of the foregoing examples describing methods of nucleic acid sequencing, the nucleic acid analyte may be DNA and the method determines the sequence of the DNA molecule. In one example, the DNA is genomic DNA. In another example, the DNA is complementary DNA (cDNA) which has been derived from an RNA. In accordance with examples in which the nucleic acid analyte is cDNA, the method may comprise determining the sequence of RNA based on the sequence for the corresponding cDNA.

[0080] In accordance with examples in which the method of sequencing a nucleic acid molecule determines the sequence of DNA or RNA as described herein, the nucleic acid polymerase may be a DNA polymerase.

[0081] In another example, the nucleic acid analyte may be an RNA molecule and the nucleic acid polymerase is a reverse transcriptase. In accordance with this example, the method may determine the sequence of the RNA molecule directly by detecting the order of enhanced fluorescence emission signatures as the labelled nucleotides sequentially bind to, and form ternary complexes with, complementary ribonucleotides within the RNA molecule and theAtorney Docket No.: 67649-702601 reverse transcriptase, during synthesis of a nascent cDNA strand which is complementary to the RNA molecule. Determining the sequence of an RNA molecule may employ a SBS method as described herein, wherein the sequence of the RNA molecule is determined by determining the order of enhanced distinct fluorescence emission signatures as the labelled nucleotides are bound to complementary ribonucleotides within the active site of the reverse transcriptase and sequentially incorporated into the nascent cDNA strand which is complementary to the RNA molecule. Alternatively, determining the sequence of an RNA molecule may employ a SBB method as described herein, wherein the sequence of the RNA molecule is determined by determining the order of enhanced distinct fluorescence emission signatures from labelled nucleotides as they sequentially bind to, and form ternary complexes with, complementary ribonucleotides within the RNA molecule and the reverse transcriptase, during synthesis of the nascent cDNA strand which is complementary to the RNA molecule.

[0082] In each of the foregoing examples describing methods of nucleic acid sequencing, the nucleotide of the labelled nucleotides may be further selected from a synthetic nucleotide. In one example, the synthetic nucleotide is selected from 5-methylcytidine, N-methylcytidine, and N6-methyladenosine.

[0083] In each of the foregoing examples describing methods of nucleic acid sequencing, the fluorophore of the labelled nucleotide may be directly or indirectly linked to the nucleotide via a 5’ phosphate or 5’ polyphosphate of the nucleotide.

[0084] For those examples describing methods of nucleic acid sequencing where the labelled nucleotides comprise both the fluorophore and the quencher (z.e., “dual -labelled nucleotides”), the fluorophore and quencher may both be indirectly linked to the nucleotide the via a 5’ phosphate or 5’ polyphosphate of the nucleotide. The polyphosphate may be a tri-, tetra-, penta- or hexa-phosphate. In one example, the polyphosphate is a tri-phosphate. In one example, the polyphosphate is a tetra-phosphate. In one example, the polyphosphate is a penta-phosphate. In one example, the polyphosphate is a hexa-phosphate.

[0085] In some examples, the fluorophore is bound to the terminal phosphate of the polyphosphate.

[0086] For those examples describing methods of nucleic acid sequencing where the labelled nucleotides comprise both the fluorophore and the quencher (i.e., ‘dual -lab el led nucleotides’), the dual-labelled nucleotide may be a compound of formula (I):A1 „R1 „A2.„R2 „.A3Atorney Docket No.: 67649-702601(Formula I) wherein:R1is or comprises the fluorophore;R2is or comprises the quencher;Ai ,a 3u * • h"t3"X I .A and A are each independently absent or is « or * L3-“X--L4;A2is absentL1to L3are each independently absent or a divalent linking moiety which is uninterrupted or interrupted, and optionally substituted;L4is absent or is a terminal moiety which is uninterrupted or interrupted, and optionally substituted;L5is absent or is a trivalent linking moiety which is uninterrupted or interrupted, and optionally substituted; each X is independently absent or is a nucleotide, on the proviso that at least one X is present; and wherein R1and R2are optionally interchangeable.

[0087] In some examples, the dual-labelled nucleotide is a compound of any one of formulae (II) to (IV):Formula (II)Formula (III)Formula (IV) wherein X is a nucleotide.Atorney Docket No.: 67649-702601

[0088] In some examples, the dual-labelled nucleotide is a compound of any one of formula (Ila)-(IIf):Formula (lie) Formula (lid)Formula (lie) Formula (Ilf) wherein:B1is a nucleobase; n is 1 to 5;R3is H, Me, an alcohol protecting group, or a 3’ blocker; andR9is H, OH, or OMe.

[0089] In some examples, the dual-labelled nucleotide is a compound of any one of formula (Illa)-(IIId):Atorney Docket No.: 67649-702601Formula (IIIc) Formula (Hid) wherein:B1is a nucleobase; n is 1 to 5;R3is H, Me, an alcohol protecting group, or a 3’ blocker; andR9is H, OH, or OMe.

[0090] In some examples, the dual labelled nucleotide is a compound of any one of formula (IVa)-(IVd):Formula (IVa) Formula (IVb)Atorney Docket No.: 67649-702601Formula (IVc) Formula (IVd) wherein:B1is a nucleobase; n is 1 to 5;R3is H, Me, an alcohol protecting group, or a 3’ blocker; andR9is H, OH, or OMe.

[0091] In each of the forgoing examples described dual-labelled nucleotides, L1toL5are each independently absent or an aliphatic group which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, -C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, - S(=O)2-, -N(R4)C(=S)N(R4)-, and -N(R4)C(=O)N(R4)-, and is optionally substituted with one or more R5, wherein: each R4is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, alkylcarbocyclyl, and alkylheterocyclyl, each of which is optionally substituted with one or more R5, each R5is independently selected from the group consisting of H, halogen, Ci-ioalkyl, OCi-ioalkyl, Ci-iohaloalkyl, OCi-iohaloalkyl, C2-ioalkenyl, C2-ioalkynyl, OC2-ioalkenyl, OC2- walkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, Ci-ioalkyl-3-10- membered-carbocyclyl, Ci-ioalkyl-3-10-membered-heterocyclyl, -NO2, -CN, -SCN, -N3, =0, -N(R5)2, -C(=O)N(R5)2, -S(=O)N(R5)2, -S(=O)2N(R5)2, -OR5, -SR5, OC(=O)R5, -C(=O)R5, - C(=O)OR5-S(=O)R5, -S(=O)2R5, -S(=O)OR5, -S(=O)2OR5, -S(=O)(OR5)2, -OS(=O)R5, - OS(=O)2R5, -OS(=O)OR9, -OS(=O)2OR5, -OS(=O)(OR5)2, -N(R5)C(=O)R5, -N(R5)S(=O)R5, N(R5)C(=O)N(R5)2, -N(R5)S(=O)2R5, -P(=O)(OR5)2, -P(=O)OR5(R5), -P(=O)(R5)2, - OP(=O)(OR5)2, -OP(=O)OR5(R5) and -OP(=O)(R5)2, wherein each Ci-ioalkyl, Ci-iohaloalkyl, C2-ioalkenyl, C2-ioalkynyl, 3-10 membered-carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R6;Atorney Docket No.: 67649-702601 each R6is independently selected from the group consisting of H, Ci-ealkyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, Ci-6alkyl-3-10-membered-carbocyclyl, and Ci-6alkyl-3-10-membered-heterocyclyl; wherein each Ci-ealkyl, 3-10-membered- carbocyclyl, and 3-10-membered heterocyclyl is optionally substituted with one or more R7; each R7is independently selected from the group consisting of H, halogen, -NO2, - N(R8)2, -CN, -SCN, -N3, =0, -C(=0)R8, -C(=0)0R8, -C(=O)N(R8)2, -N(R8)C(=O)R8, -OR8, -P(=O)(OR8)2, -P(=O)OR8(R8), -P(=O)(R8)2, Ci-6alkyl, and -OCi-6alkyl; and each R8is independently selected from the group consisting of H, Ci-ioalkyl, Ci-ioalkyl, C2-ioalkenyl, C2-ioalkynyl, 3-10 membered carbocyclyl, 3-10 membered heterocyclyl, Ci- ioalkyl-3- 10-membered carbocyclyl, Ci-ioalkyl-3-lO-membered heterocyclyl.

[0092] In some examples, L1to L5are each independently absent or is Ci-3oalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, - C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and - N(R4)C(=O)N(R4)-, and is optionally substituted with one or more R5.

[0093] In some examples, L1to L5are each independently absent or is Ci-2oalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, - C(=O)NH-, -C(=O)O-.

[0094] In certain examples, the method of detecting binding of a labelled species to an analyte of interest as described herein is a method of sequencing a polypeptide. According to one example in which the method described herein is a method of sequencing a polypeptide: the analyte of interest is a polypeptide analyte which is positioned within the area of electrical field enhancement, each labelled species is a labelled amino acid recognition molecule which is capable of selectively associating with at least one type of terminal amino acid of the polypeptide, and the labelled amino acid recognition molecule comprises the fluorophore and the one or more quenchers, step (I) comprises contacting the polypeptide analyte with the one or more labelled amino acid recognition molecules and a cleaving reagent for a time and under conditions such that at least one labelled amino acid recognition molecule selectively associates with a terminal amino acid of the polypeptide in the presence of the cleavage reagent, wherein selective association of the one or more labelled amino acid recognition molecules with the terminal amino acid and subsequent cleavage of the terminal amino acid from the polypeptide analyte, results in a distinct fluorescenceAtorney Docket No.: 67649-702601 emission signature for each type of terminal amino acid when the fluorophore to which the respective amino acid recognition molecule is linked is excited, wherein the distinct fluorescence emission signature for each type of terminal amino acid is enhanced within the area of electrical field enhancement, and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the terminal amino acid when selectively associated with its cognate amino acid recognition molecule and subsequently cleaved from the polypeptide terminus, and determining the identity of the terminal amino acid based on its enhanced distinct fluorescence emission signature, wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the polypeptide analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the amino acid types exposed at the polypeptide terminus are sequentially cleaved from the polypeptide by the cleavage reagent, thereby providing the sequence of the amino acid types in the polypeptide analyte.

[0095] According to another example in which the method described herein is a method of sequencing a polypeptide: the analyte of interest is a polypeptide analyte which is positioned within the area of electrical field enhancement, each labelled species is a labelled amino acid recognition molecule which is capable of selectively associating with at least one type of terminal amino acid of the polypeptide, wherein the labelled amino acid recognition molecule comprises the fluorophore, step (I) comprises contacting the polypeptide analyte with one or more of the labelled amino acid recognition molecule(s), a cleaving reagent and one or more quenchers for a time and under conditions such that at least one labelled amino acid recognition molecule selectively associates with a terminal amino acid of the polypeptide in the presence of the cleavage reagent and the one or more quenchers, wherein the one or more quenchers are not attached to the amino acid recognition molecule(s) and are provided at a concentration that attenuates fluorescence emission intensity of the fluorophore(s) when the or each fluorophore is excited, wherein selective association of the one or more labelled amino acid recognition molecules with the terminal amino acid and subsequent cleavage of the terminal amino acid from the polypeptide analyte, results in a distinct fluorescenceAtorney Docket No.: 67649-702601 emission signature for each type of terminal amino acid when the fluorophore to which the respective amino acid recognition molecule is linked is excited, wherein the distinct fluorescence emission signature for each type of terminal amino acid is enhanced within the area of electrical field enhancement, and step (II) comprises detecting the order of enhanced distinct fluorescence emission signature of the terminal amino acid when selectively associated with its cognate amino acid recognition molecule and subsequently cleaved from the polypeptide terminus, and determining the identity of the terminal amino acid based on its enhanced distinct fluorescence emission signature, wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the polypeptide analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the amino acid types exposed at the polypeptide terminus are sequentially cleaved from the polypeptide by the cleavage reagent, thereby providing the sequence of the amino acid types in the polypeptide analyte.

[0096] In accordance with examples of sequencing a polypeptide, the area of electrical field enhancement may be produced by any means in the art suitable for polypeptide sequencing, examples of which are described herein. For example, the area of electrical field enhancement may be produced by nanoparticles (such as, for example, a single nanoparticle or multiple nanoparticles, including multiple nanoparticle structures), surface structures (such as nanostructured surfaces), nanophotonic confinement structure (such as zero-mode waveguides or and microcavities) and combinations thereof.

[0097] In one example, the area of electrical field enhancement is produced by a nanophotonic confinement structure. For example, the nanophotonic confinement structure may be a zeromode waveguide (ZMW). In accordance with this example, the polypeptide analyte to be sequenced is immobilised within the ZMW.

[0098] In another example, the area of electrical field enhancement is produced by one or more plasmonic nanoparticles. Exemplary means of producing areas of electrical field enhancement using plasmonic nanoparticles, including the production of plasmonic hotspots using plasmonic nanoparticles positioned on plasmonic nanoantennas, are described hereinabove and shall be taken to apply mutatis mutandis to each and every example describing the method of sequencing a nucleic acid unless stated otherwise.

[0099] In certain examples, the area of electrical field enhancement is produced by a plasmonic nanoantenna as described herein, wherein the polypeptide analyte to be sequencedAtorney Docket No.: 67649-702601 is bound to the nanoscopic nucleic acid scaffold of the plasmonic nanoantenna and is positioned within the region unoccupied by the nanoscopic nucleic acid scaffold.

[0100] In one example, the polypeptide analyte to be sequenced is bound to the nanoscopic nucleic acid scaffold. In some examples, the polypeptide analyte is bound non-covalently to the nanoscopic nucleic acid scaffold, preferably through hydrogen bonding.|0101] In accordance with examples in which a plasmonic nanoantenna described herein is used for polypeptide sequencing, the region of the plasmonic hotspot unoccupied by the nanoscopic nucleic acid scaffold will be sufficiently large to accommodate the polypeptide analyte.

[0102] In some examples, the polypeptide analyte is bound to, or immobilized on, the nanoscopic nucleic acid scaffold through a nucleic acid linker. In some embodiments, the nucleic acid linker is double-stranded and comprises: (i) a polynucleotide which is covalently or non-covalently bound to the polypeptide analyte via an amino acid within the polypeptide analyte, and (ii) a polynucleotide forming part of, or extending from, the nanoscopic nucleic acid scaffold, wherein the polynucleotides at (i) and (ii) comprise single-stranded DNA sequences which are complementary or substantially complementary and are capable of hybridizing to one another. In one example, the polynucleotide at (i) comprises a peptide tag which is bound to polypeptide analyte via an isopeptide bond formed between an amino acid within the polypeptide analyte and an amino acid within the peptide tag. In one example, the nucleic acid linker is bound to the polypeptide analyte via an amino acid at the terminus of the polypeptide, preferably the terminus which is not being targeted for Edman degradation by the labelled amino acid recognition molecules in the presence of the cleavage reagent. For example, the polypeptide analyte may comprise a peptide tag (e.g., a universal peptide tag) at its terminus, and the nucleic acid linker may be bound to the polypeptide analyte via an amino acid in the peptide tag at the terminus of the polypeptide analyte. The peptide tag (e.g., universal peptide tag) may be conjugated to the terminus of the polypetide analyte.

[0103] Each of the polynucleotides of the nucleic acid linker which binds or immobilises the polypeptide analyte on the nanoscopic nucleic acid scaffold comprises a region of sufficient length and complementarity to hybridise to one another. In one example, each polynucleotide of the nucleic acid linker comprises about 20 nucleotides to about 50 nucleotides. In another example, each polynucleotide of the nucleic acid linker comprises about 20 nucleotides to about 30 nucleotides. In each of these examples, the polynucleotides of the nucleic acid linkerAtorney Docket No.: 67649-702601 each comprise a region of about 10 to about 25 nucleotides in length which are complementarity and capable of hybridizing to one another.

[0104] In accordance with examples where method is for polypeptide sequencing, the labelled amino acid recognition molecule and the cleavage reagent are the same molecule (e.g., a single molecule comprising an amino acid recognition moiety and a cleavage reagent). In other examples, the labelled amino acid recognition molecule and the cleavage reagent are different molecules. In each of these examples, the cleavage reagent may be phenylisothiocyanate (PTC).

[0105] In some examples, one or more of the labelled amino acid recognition molecules is capable of reversibly associating with the terminal amino acid of the polypeptide. In some examples, each of the labelled amino acid recognition molecules are capable of reversibly associating with the terminal amino acid of the polypeptide.

[0106] In some examples, one or more of the labelled amino acid recognition molecules are capable of binding selectively and with affinity (KD) to one type of amino acid. In some examples, each of the labelled amino acid recognition molecules is capable of binding selectively and with affinity (KD) to one type of amino acid. For example, the binding affinity of the or each labelled amino acid recognition molecule for its cognate type of amino acid is at least about 50 nM or higher. For example, the binding affinity of the or each labelled amino acid recognition molecule for its cognate type of amino acid may be between about 50 nM and about 50 gM, such as between about 100 nM and about 10 pM, or between about 500 nM and about 50 gM.

[0107] As describes herein, each labelled amino acid recognition molecule selectively binds to one type of amino acid. In one example, the one type of amino acid is an amino-terminal amino acid. In one example, the one type of amino acid is a carboxy-terminal amino acid. In one example, the one type of amino acid is one of the twenty naturally-occurring amino acids or a. subset of types thereof. In one example, the one type of amino acid is a modified variant of one of the twenty naturally occurring amino acids or a subset of unmodified and / or modifi ed variants thereof In one example, the one type of amino acid is selected from amino acids with charged side chains (e.g, positively and / or negatively charged side chains), amino acids with polar side chains (e.g.. polar uncharged side chains), amino acids with nonpolar side chains (e.g, nonpolar aliphatic and / or aromatic side chains), and amino acids with hydrophobic side chains.Atorney Docket No.: 67649-702601

[0108] In some examples of the method of sequencing a polypeptide, the polypeptide analyte is contacted with a plurality of labelled amino acid recognition molecules, each selectively associating with a different type of amino acid as described herein and each having a distinct fluorescence emission signature. Each of the distinct fluorescence emission signatures may be distinguished from one another based on one or more of the following characteristics: peak emission wavelength, fluorescence emission intensity, fluorescence emission lifetime, duration of fluorescence, duration between successive fluorescence emissions, or any combinations thereof.

[0109] In one example, each labelled amino acid recognition molecule which selectively associates with a distinct type of amino acid is linked to a fluorophore having a distinct fluorescence emission signature which can be distinguished from fluorescence emission signatures of other labelled amino acid recognition molecules. For example, each labelled amino acid recognition molecule which selectively associates with a distinct type of amino acid may be linked to a fluorophore having a distinct peak emission wavelength.

[0110] In certain examples, the method of detecting binding of a labelled species to an analyte of interest as described herein is a method of biosensing (e.g., to detect the presence or absence of an analyte of interest in a test sample). According to one example in which the method described herein is a method of biosensing: step (I) comprises contacting a capture reagent positioned within the area of electrical field enhancement with the sample and the labelled species, wherein the capture reagent and the labelled species are each independently capable of binding to the analyte of interest, the fluorophore and quencher are positioned relative to one another on the labelled species such that the quencher attenuates the fluorophore’ s emission intensity when the fluorophore is excited irrespective of structural conformation of the labelled species, and detecting the enhanced distinct fluorescence emission signature of the labelled species at step (II) indicates that the labelled species is bound to the analyte of interest, indicating the presence of the analyte of interest in the sample.

[0111] In some examples, the method of biosensing is capable of sensing for two or more analytes of interest, wherein: step (I) comprises contacting the sample with two or more sets of capture reagent and labelled species, wherein the capture reagent of each set is positioned within the area of electrical field enhancement, the labelled species of each set has a distinct fluorescence emission signature when its fluorophore is excited, the capture reagent and the labelled speciesAtorney Docket No.: 67649-702601 of a respective set are capable of independently binding to the same analyte of interest, and each set of capture reagent and labelled species binds to a different analyte of interest; and detecting the enhanced distinct fluorescence emission signature of the labelled species of a set at step (II) indicates the presence of the respective analyte of interest in the sample. In some examples, the method comprises detecting multiple different analytes of interest in the sample.

[0112] In each example describing a method of biosensing, the or each labelled species has distinct fluorescence emission signature which is enhanced when the fluorophore is excited within the area of electrical field enhancement. As described herein, distinct fluorescence emission signatures of the labelled species may be distinguishable based on one or more of the following characteristics: peak emission wavelength, fluorescence emission intensity, fluorescence emission lifetime, duration between successive fluorescence emissions, or any combinations thereof.

[0113] In accordance with examples in which enhanced distinct fluorescence emission signature are detected for multiple labelled species (e.g., when biosensing for multiple analytes of interest), the respective labelled species may be distinguished from one another based on differences in one or more of the following: peak emission wavelength, fluorescence emission intensity, fluorescence emission lifetime, duration between successive fluorescence emissions, or any combinations thereof. For example, where the method comprises detecting enhanced distinct fluorescence emission signatures for multiple labelled species, at least one or each of the labelled species is attached to a fluorophore having a distinct peak emission wavelength. For example, the distinct peak emission wavelengths of the multiple labelled species may be separated by 10 nm or more relative to each other.

[0114] In some examples, two or more labelled species (i.e., in different sets) comprise the same fluorophore, and the enhanced distinct fluorescence emission signatures of the respective labelled species are distinguished based on differences in fluorescence emission intensity and / or fluorescence emission lifetime.

[0115] According to certain examples in which the method is for biosensing an analyte of interest in a sample, the labelled species may be a compound of formula (I):A1™R1„A2™R2™A3(Formula I) wherein:Atorney Docket No.: 67649-702601R1is or comprises the fluorophore;R2is or comprises the quencher;A1and A3are each independently absent or is;A2is absentL1to L3are each independently absent or a divalent linking moiety which is uninterrupted or interrupted, and optionally substituted;L4is a terminal moiety which is uninterrupted or interrupted, and optionally substituted;L5is a trivalent linking moiety which is uninterrupted or interrupted, and optionally substituted; each X is independently absent or comprises an analyte binding moiety, on the proviso that at least one X is present; and wherein R1and R2are interchangeable.

[0116] In some examples, the labelled species of formula (I) is a compound of any one of formulae (II) to (IV):R^~~L1~~X~'L2~“RaFormula (II)X~L3~R1“L1~~RZFormula (III)Formula (IV), wherein X is present and comprises the analyte binding moiety. In one example, the labelled species is a compound of formulae (II):R . L1-X-L2. R2Formula (II)Atorney Docket No.: 67649-702601 wherein X is present and comprises the analyte binding moiety. In accordance with one example in which the labelled species is a compound of formulae (II), L1and L2is absent, and X is a nucleic acid sequence comprising and / or consisting of 1 to 30 contiguous nucleotides. In accordance with this example, X may be a polynucleotide sequence consisting of 1 to 20 contiguous nucleotides. For example, X may be a polynucleotide sequence consisting of 1 to 10 contiguous nucleotides (e.g., 1, or, 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 contiguous nucleotides). For example, X may be a polynucleotide sequence consisting of 10 to 20 contiguous nucleotides (e.g., 10, or 11, or, 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 contiguous nucleotides).

[0117] In some examples, the labelled species is a compound of formulae (III):X~L3”R1"~L1~~R2Formula (III), wherein X is present and comprises the analyte binding moiety. In accordance with an example in which the labelled species is a compound of formulae (III), L3may be absent. Alternatively, or in addition, LI of formulae (III) may be a nucleic acid sequence comprising and / or consisting of 1 to 30 contiguous nucleotides. In accordance with this example, LI of formulae (III) may be a polynucleotide sequence consisting of 1 to 20 contiguous nucleotides. For example, LI of formulae (III) may be a polynucleotide sequence consisting of 1 to 10 contiguous nucleotides (e.g., 1, or, 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 contiguous nucleotides). For example, LI of formulae (III) may be a polynucleotide sequence consisting of 10 to 20 contiguous nucleotides (e.g., 10, or 11, or, 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 contiguous nucleotides).

[0118] In each of the foregoing examples describing labelled species which is a compound of any one of formulae (II) to (IV), R1may be a labelled nucleotide comprising a nucleotide covalently linked to the quencher, wherein the nucleotide is selected from the group consisting of A, G, T or C.

[0119] According to any one or more of the foregoing examples describing a method for biosensing an analyte of interest in a sample, the analyte binding moiety may be selected from the group consisting of: small molecule, peptide, polypeptide, protein and nucleic acid.

[0120] In one example, the analyte binding moiety is a protein or peptide. For example, the analyte binding moiety may be an antigen-binding protein (e.g., an antibody or an antigenAtorney Docket No.: 67649-702601 binding fragment thereof), antigen or antigenic fragment. Exemplary antibody formats or antigen binding fragments which may be used as the analyte binding moiety include, but is not limited to, a full length (‘intact’) antibody, a Fab, a F(ab')2, an Fv, a single chain antibody (SCA), diabody, triabody or tetrabody. In other examples, the analyte binding moiety is a peptide (e.g., a peptide- or protein-binding peptide).|0121] In other examples, the analyte binding moiety is a nucleic acid sequence comprising and / or consisting of 1 to 100 nucleotides. For example, the analyte binding moiety may be a polynucleotide comprising and / or consisting a sequence of about 8 to 50 contiguous nucleotides (e.g., about 10 to about 30 contiguous nucleotides, or about 15 to about 25 contiguous nucleotides, or about 20 contiguous nucleotides) which is complementary or substantially complementary to a polynucleotide sequence in the analyte of interest such that it is capable of hybridising thereto. The analyte binding moiety may comprise a single polynucleotide sequence which is complementary or substantially complementary to a polynucleotide sequence in the analyte of interest such that it is capable of hybridising thereto. Alternatively, the analyte binding moiety may comprise a plurality of polynucleotide sequences which are each complementary or substantially complementary to a polynucleotide sequence in the analyte of interest, and capable of hybridising thereto.

[0122] In some examples, the labelled species is a compound of formula (I) as described herein, wherein L1to L5are each independently absent or an aliphatic group which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, - C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and - N(R4)C(=O)N(R4)-, and is optionally substituted with one or more R5, wherein: each R4is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, alkylcarbocyclyl, and alkylheterocyclyl, each of which is optionally substituted with one or more R5, each R5is independently selected from the group consisting of H, halogen, Ci- walkyl, OCi-ioalkyl, Ci-iohaloalkyl, OCi-iohaloalkyl, C2-ioalkenyl, C2-ioalkynyl, OC2- walkenyl, OC2-ioalkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, Ci-ioalkyl-3-10-membered-carbocyclyl, Ci-ioalkyl-3-10-membered-heterocyclyl, - NO2, -CN, -SCN, -N3, =0, -N(R5)2, -C(=O)N(R5)2, -S(=O)N(R5)2, -S(=O)2N(R5)2, - OR5, -SR5, OC(=O)R5, -C(=O)R5, -C(=O)OR5-S(=O)R5, -S(=O)2R5, -S(=O)OR5, - S(=O)2OR5, -S(=O)(OR5)2, -OS(=O)R5, -OS(=O)2R5, -OS(=O)OR9, -OS(=O)2OR5, - OS(=O)(OR5)2, -N(R5)C(=O)R5, -N(R5)S(=O)R5, N(R5)C(=O)N(R5)2, -Atorney Docket No.: 67649-702601N(R5)S(=O)2R5, -P(=O)(OR5)2, -P(=O)OR5(R5), -P(=O)(R5)2, -OP(=O)(OR5)2, - OP(=O)OR5(R5) and -OP(=O)(R5)2, wherein each Ci-ioalkyl, Ci-iohaloalkyl, C2. walkenyl, C2-ioalkynyl, 3-10 membered-carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R6; each R6is independently selected from the group consisting of H, Ci-ealkyl, 3- 10 membered carbocyclyl, 3-10-membered heterocyclyl, Ci-6alkyl-3-10-membered- carbocyclyl, and Ci-6alkyl-3-10-membered-heterocyclyl; wherein each Ci-ealkyl, 3-10- membered-carbocyclyl, and 3-10-membered heterocyclyl is optionally substituted with one or more R7; each R7is independently selected from the group consisting of H, halogen, -NO2, -N(R8)2, -CN, -SCN, -N3, =0, -C(=O)R8, -C(=O)OR8, -C(=O)N(R8)2, -N(R8)C(=O)R8, -OR8, -P(=O)(OR8)2, -P(=O)OR8(R8), -P(=O)(R8)2, Ci-6alkyl, and -OCi-6alkyl, each R8is independently selected from the group consisting of H, Ci-ioalkyl, Ci- walkyl, C2-ioalkenyl, C2-ioalkynyl, 3-10 membered carbocyclyl, 3-10 membered heterocyclyl, Ci-ioalkyl-3-lO-membered carbocyclyl, Ci-ioalkyl-3-lO-membered heterocyclyl.

[0123] In some examples, the labelled species is a compound of formula (I) as described herein, wherein L1to L5are each independently absent or is Ci-3oalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, -C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and -N(R4)C(=O)N(R4)-, and is optionally substituted with one or more R5.

[0124] In some examples, the labelled species is a compound of formula (I) as described herein, wherein L1to L5are each independently absent or is Ci-2oalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, -C(=O)NH-, -C(=O)O-Also provided herein is a compound of formula (I):(Formula I) wherein:R1is or comprises a fluorophore having a peak emission wavelength of X when excited;Atorney Docket No.: 67649-702601R2is or comprises a quencher having an emission spectrum which is capable of attenuating intensity of the peak emission wavelength of the fluorophore at R1;A1and A3are each independently absent or is;A2is absentL1to L3are each independently absent or a divalent linking moiety which is uninterrupted or interrupted, and optionally substituted;L4is a terminal moiety which is uninterrupted or interrupted, and optionally substituted;L5is a trivalent linking moiety which is uninterrupted or interrupted, and optionally substituted; each X is independently absent or comprises an analyte binding moiety, on the proviso that at least one X is present; wherein R1and R2are interchangeable, and wherein the fluorophore and quencher are positioned relative to one another on the compound such that the quencher attenuates peak emission wavelength of the fluorophore irrespective of the compound conformation.

[0125] Exemplary compounds of formula (I), including substructures of formula (I) selected from the compounds of formulas (II), (III) and (IV), are described hereinabove in the context of methods of biosensing and shall be taken to apply mutatis mutandis to each and every example describing compounds per se unless stated otherwise.

[0126] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.Brief description of the drawings

[0127] Figure 1 illustrates noise values calculated (standard deviation of a robust Gaussian fit (least absolute residuals) from extracted trace of individual enhancement regions in a DNA- PAINT like transient binding assay.Atorney Docket No.: 67649-702601

[0128] Figure 2 illustrates the signal-to-noise ratio vs proportion of particles, for a DNA- PAINT like transient binding assay.Detailed DescriptionGeneral

[0129] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, feature, composition of matter, group of steps or group of features or compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, features, compositions of matter, groups of steps or groups of features or compositions of matter.

[0130] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0131] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0132] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

[0133] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0134] Unless otherwise indicated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub.Atorney Docket No.: 67649-702601Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0135] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0136] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0137] Each feature of any particular aspect or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment of the present disclosure.

[0138] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0139] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", is understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0140] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.Specific terms

[0141] The compounds of the present disclosure may contain chiral (asymmetric) centers or the molecule as a whole may be chiral. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present disclosure.

[0142] The term “halo” or “halogen” whether employed alone or in compound words such as haloalkyl, represents fluorine, chlorine, bromine or iodine. Further, when used in compoundAtorney Docket No.: 67649-702601 words such as haloalkyl, the alkyl may be partially halogenated or fully substituted with halogen atoms which may be independently the same or different. Examples of haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups. Further examples of haloalkyl groups include -CF3, -CCI3, and -CH2CF3, -CF2CF3 and CH2CHFCI.|0143] As used herein, the term “alkyl” whether used alone, or in compound words such as haloalkyl, cycloalkyl, alkylcycloalkyl, alkylcarbocyclyl, heteroalkyl, alkylheterocyclyl, alkylheteroaryl, alkylamide, alkylphosphonate and alkylaryl, represents straight chain (i.e. linear) or branched chain hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl groups. In one example, the alkyl group is of 1 to 20 carbon atoms (i.e. Ci- 2oalkyl). In another examples, the alkyl is a group of 1 to 10 carbon atoms (i.e. Ci-ioalkyl). In another example, the alkyl group is of 1 to 6 carbon atoms (i.e. Ci-ealkyl).

[0144] As used herein, the term “heteroalkyl” represents straight chain (i.e. linear) or branched chain hydrocarbon groups which are analogous to an alkyl group, but in which one or more carbon atoms is / are replaced by one or more heteroatoms selected from nitrogen, sulfur, and oxygen.

[0145] As used herein, the term “alkenyl” represents straight (i.e. linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon double bond. Examples of alkenyl groups include ethylene, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl groups. In one example, the alkenyl group is of 2 to 20 carbon atoms (i.e. C2-2oalkenyl). In another example, the alkenyl is a group 2 to 10 carbon atoms (i.e. C2-ioalkenyl). In another example, the alkenyl group is of 2 to 6 carbon atoms (i.e. C2- ealkenyl).

[0146] As used herein, the term “alkynyl” represents straight (i.e. linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon triple bond. Examples of alkenyl groups include, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl groups. In one example, the alkynyl group is of 2 to 20 carbon atoms (i.e. C2-2oalkynyl). In one example, the alkynyl group is of 2 to 10 carbon atoms (i.e. C2- walkynyl). In another examples, the alkynyl group is of 2 to 6 carbon atoms (i.e. C2-ealkynyl).

[0147] As used herein, the term “haloalkyl” represents to an alkyl group having at least one halogen substituent, where “alkyl” and “halogen” are as described above. For example, the haloalkyl group may have at least one, two or three halogen substituents. Examples ofAtorney Docket No.: 67649-702601 haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups. Further examples of haloalkyl groups include -CF3, -CCI3, and -CH2CF3, -CF2CF3 and CH2CHFCI. In one example, the haloalkyl group is of 1 to 20 carbon atoms i.e. Ci- 2ohaloalkyl). In one example, the haloalkyl group is of 1 to 10 carbon atoms i.e. Ci- lohaloalkyl). In another example, the haloalkyl group is of 1 to 6 carbon atoms i.e. Ci- ehaloalkyl).

[0148] As used herein, the terms “carbocyclyl” and “carbocycle” whether used alone, or in compound words such as alkylcarbocyclyl, represents a monocyclic or polycyclic ring system wherein the ring atoms are all carbon atoms, e.g., of about 3 to about 20 carbon atoms, and which may be aromatic, non-aromatic, saturated, or unsaturated, and may be substituted and / or contain fused rings. In one example, the carbocyclyl group is of 3 to 20 carbon atoms i.e. C3-2o-membered carbocyclyl). In another example, the carbocyclyl group is of 3 to 10 carbon atoms i.e. Cs-io-membered carbocyclyl). Examples of such groups include aryl groups such as phenyl, naphthyl, anthracenyl or fluorenyl, saturated groups such as cycloalkyl and cycloalkenyl groups e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl groups, or fully or partially hydrogenated phenyl, naphthyl and fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0149] As used herein, the term “cycloalkyl” whether used alone, or in compound words such as alkylcycloalkyl, refers to a monocyclic or polycyclic carbocyclic ring system of varying sizes, e.g., from about 3 to about 20 carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0150] As used herein, the term “heterocyclyl” whether used alone or in compound words such as alkylheterocyclyl, refers to a monocyclic or polycyclic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and wherein the ring system may be aromatic such as a “heteroaryl” group, non-aromatic, saturated, or unsaturated, and may be substituted and / or contain fused rings. Heterocyclyl groups containing a suitable nitrogen atom include the corresponding N-oxides. In one example, the heterocyclyl group is of 3 to 20 atoms (i.e. 3-20-membered heterocyclyl). In another example, the heterocyclyl group is of 3 to 10 atoms (i.e. 3-10-membered heterocyclyl). The heteroatom may preferably be N, O or S. ExamplesAtorney Docket No.: 67649-702601 of monocyclic non-aromatic heterocyclyl groups include aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl and azepanyl. Examples of bicyclic heterocyclyl groups in which one of the rings is non-aromatic include dihydrobenzofuranyl, indanyl, indolinyl, isoindolinyl, tetrahydroisoquinolinyl, tetrahydroquinolyl, and benzoazepanyl. Examples of monocyclic aromatic heterocyclyl groups (also referred to as monocyclic heteroaryl groups) include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl (e.g. the radical derived from pyridine), triazolyl, triazinyl, pyridazyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic aromatic heterocyclyl groups (also referred to as bicyclic heteroaryl groups) include quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0151] Amino acids may be referred to herein by their full name, three letter code, or single letter code, all of which will be understood by the person skilled in the art. Likewise, nucleosides may be referred to by their full name, or single letter code, either of which will be understood by the person skilled in the art.

[0152] As will be understood, an “aromatic” group means a cyclic group having 4m+2 i electrons, where m is an integer equal to or greater than 1. As used herein, “aromatic” is used interchangeably with “aryl” to refer to an aromatic group, regardless of the valency of aromatic group.

[0153] As used herein, the term “aryl” whether used alone, or in compound words such as alkylaryl, represents a monocyclic (e.g. phenyl) or polycyclic (e.g. naphthyl) aromatic carbocyclic ring system. In one example, the aryl group is of 3 to 20 carbon atoms (i.e., an aromatic 3-20 membered carbocyclyl). In another example, the aryl group is of 3 to 10 carbon atoms (i.e., an aromatic 3-10 membered carbocyclyl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl or fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems. Related to the term aryl, the term “aralkyl” as used herein refers to an alkyl group wherein a hydrogen atom is replaced by an aryl group as a substituent. Examples of alkylaryl groups include, but are not limited to, an optionally substituted benzyl (e.g. CEE-phenyl).Atorney Docket No.: 67649-702601

[0154] As used herein, the term “heteroaryl” whether used alone, or in compound words such as alkylheteroaryl, represents a monocyclic or polycyclic aromatic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and may be substituted and / or contain fused rings. Heteroaryl groups containing a suitable nitrogen atom include the corresponding N- oxides. In one example, the heteroaryl group is of 3 to 20 atoms (i.e. 3-20-membered heteroaryl). In another example, the heteroaryl group is of 3 to 10 atoms (i.e. 3-10-membered heteroaryl). Examples of monocyclic heteroaryl groups include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic heteroaryl groups include quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. All regioisomers are contemplated, e.g. 2-pyridyl, 3-pyridyl and 4- pyridyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0155] As used herein, the term “divalent linking moiety” refers to any divalent group capable of linking, joining, bonding or attaching two chemical moi eties. As used herein, the term “trivalent linking moiety” refers to any trivalent group capable of linking, joining, bonding or attaching three chemical moieties, one of which may be an analyte binding group. As used herein, the term “terminal moiety” refers to a monovalent group capable of joining, bonding or attaching to the end of a chemical moiety.“-C(=O)-” represents a carbonyl linker group.“-C(=O)O-” represents an ester linking group.“-C(=O)S-” represents a thioester linking group.“-C(=O)NH-” or “-C(=O)NR-” represents an amide linker group.“-S(=O)2-” represents a sulfone linker group.“-S(=O)NH-” or “-S(=O)NR-” represents a sulfmamide linker group. “-S(=O)2NH-” or “-S(=O)2NR-” represents a sulfonamide linker group. “-OS(=O)2-” represents a sulfonate ester linker group.“-O-” represents an ether linker group.“-NH-” or “-NR-” represents an amine linker group.“-S-” represents a sulfide linker.Atorney Docket No.: 67649-702601“-NHC(=S)NH-” or “-N(R)C(=S)N(R)-” represents a thiourea linker group. “-NHC(=O)NH-” or “-N(R)C(=O)N(R)-” represents a urea linking group.“-(CH2)m-“ represents an alkylene linking group, including an alkylene bridge, having a defined number (“m”) of methylene (-CH2-) units.

[0156] Unless otherwise stated or structurally depicted, it will be appreciated that the orientation of the linker groups described above and herein within the compound of Formula (1) (as well as sub-formulae thereof) are undefined. That is, the linker groups may be attached at either side within the compound of Formula (1).

[0157] As used herein, the term “saturated” refers to a group where all available valence bonds of the backbone atoms are attached to other atoms Representative examples of saturated groups include, but are not limited to, butyl, cyclohexyl, piperidine, and the like.

[0158] As used herein, the term “unsaturated” refers to a group where at least one valence bond of two adjacent backbone atoms is not attached to other atoms. Representative examples include, but are not limited to, alkenes (e.g., -CH2-CH2CFUCH), phenyl, pyrrole, and the like.

[0159] As used herein, the term “optionally substituted” means that a functional group is either substituted or unsubstituted, at any available position.

[0160] As used herein, the term “substituted” refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group (i.e., substituent).

[0161] As used herein, the term “unsubstituted” refers to a group that does not have any further groups attached thereto or substituted therefore.

[0162] As used herein, the term “stereoisomer” refers to compounds having the same molecular formula and sequence of bonded atoms (i.e., atom connectivity), though differ in the three-dimensional orientations of their atoms in space. As used herein, the term “enantiomers” refers to two compounds that are stereoisomers in that they are non- superimposable mirror images of one another. Relevant stereocenters may be denoted with (R)- or (S)- configuration.Method of detecting binding of a labelled species to an analyte of interest

[0163] As described herein, the inventors have shown for the first time that a quenched fluorescently-labelled species which is capable of binding to an analyte of interest can be used to detect that analyte when the labelled species is bound thereto in an area of electrical field enhancement when the fluorescent label is excited. Furthermore, and surprisingly, the inventors have shown that an increase in signal-to-noise ratio (SNR) can be achieved when aAtorney Docket No.: 67649-702601 fluorescently-labelled species within an enhancement region is coupled and / or in close proximity to a quencher which attenuates or reduces the light emitted by the fluorescently- labelled species. The inventors have also found that this holds true even at equivalent concentrations of fluorescent label and quencher. This discovery is counterintuitive, because quenchers are widely understood to “quench” or attenuate the fluorescence emissions from a fluorescent species when excited, and thus a reduction in the intensity of the observable fluorescence signal would be expected when the fluorophore is in proximity to a quencher. However, in contrast to the conventional wisdom, the inventors have shown experimentally, and for the first time, that the SNR associated with a binding event between a fluorescently- labelled species and an analyte of interest within in an area of electrical field enhancement is increased in the presence of a quencher, compared to the SNR associated with the same binding event in the absence of a quencher. Without being bound to any one theory, the inventors believe that this observed increase in SNR may be the result of engineering the spontaneous decay rate of the fluorophore attached to the fluorescently-labelled species in two ways at the same time. Firstly, the positioning the quencher in close proximity to the fluorophore increases the non-radiative decay rate of the fluorophore, leading to a reduction in quantum yield. Secondly, positioning of the fluorophore within an enhancement region increases the rate of radiative decay, which leads to an increase in quantum yield within the enhancement region. The inventors postulate that combining these two effects lead to reduced background noise and a recovered signal within the enhancement region, because the increase in rate of radiative decay for fluorophores within the enhancement region is relatively greater than the increase in rate of non-radiative decay for fluorophores which are quenched. This surprising finding is likely to provide a number practical advantages in the context of analyte detection assays, such as biosensing assays and DNA / protein sequencing methods, including inter alia (i) increasing efficiency for detecting fluorescent signal associated with an analyte of interest and / or binding event, (ii) improving accuracy of detection and subsequent data integrity, (iii) reducing the excitation power that may be required in a detection assay without adversely compromising fluorescent signal detection efficiency, (iv) enabling the use of less efficient and / or sophisticated equipment (e.g., optical devices) for detecting fluorescent signal, and / or (iv) allowing for increased concentration of fluorescently-labelled species in assay compositions without a corresponding increase in background noise, thereby increasing the efficiency with which the fluorescently-labelled species may bind or otherwise be used to detect an analyte.Atorney Docket No.: 67649-702601

[0164] Accordingly, the present disclosure provides a method of detecting binding of a labelled species to an analyte of interest, comprising:(I) contacting one or more labelled species, each comprising a fluorophore, with the analyte of interest in the presence of one or more quenchers of the fluorophore(s) and within an area of electrical field enhancement, wherein:(i) the or each labelled species comprises one or more quenchers, and the fluorophore and one or more quenchers are positioned relative to one another on the labelled species such that the one or more quenchers attenuate the fluorescence emission intensity of the fluorophore(s) when the or each fluorophore is excited; and / or(ii) the one or more quenchers are provided separately at a concentration that attenuates the fluorescence emission intensity of the fluorophore(s) when the or each fluorophore is excited, wherein the labelled species has a distinct fluorescence emission signature which is enhanced when the fluorophore is excited within the area of electrical field enhancement; and(II) detecting the enhanced distinct fluorescence emission signature of the labelled species within the area of electrical field enhancement following excitation of the fluorophore with light at a wavelength corresponding to an excitation wavelength of the fluorophore, wherein detection of the enhanced distinct fluorescence emission signature indicates that the labelled species is bound to the analyte of interest.

[0165] In this regard, the present method is broadly applicable to any application in which one desires to detect an analyte by illuminating a compound (e.g. a labelled species) that is bound to the analyte. The method of the disclosure may be particularly useful for analysis of individual molecules or molecular interactions and / or interactions of surface-coupled reactants, for example and without limitation, polynucleotide or polypeptide polymerization reactions, polynucleotide or polypeptide degradation or cleavage reactions (e.g. Edman degradation, protein sequencing), hybridization reactions (e.g. DNA sequencing), binding assays (e.g., biosensing), and the like. Further details regarding such single- molecule / molecular complex analyses are provided, e.g., in US12 / 413,258, US12 / 328,715,Atorney Docket No.: 67649-702601US12 / 413,226, US61 / 186,661, US20070206187, US7,056,661 and US6,917,726, the entire contents of each of which is incorporated herein by reference.

[0166] It will therefore be appreciated that there is no particular limitation as to types of applications that the method of the disclosure may be put to use, provided that the method involves detecting binding of a labelled species to an analyte of interest. In some embodiments, the method is a method of sequencing a polypeptide molecule. In some embodiments, the method is a method of sequencing a nucleic acid molecule. In some embodiments, the method is a fluorescence-based biosensing assay. These application- specific uses for the method of the disclosure are described in further detail below.

[0167] As used herein, “analyte” or “analyte of interest” refers to any molecule, compound or complex that is desired to be, or is capable of being, identified, detected, quantified, or otherwise measured directly or indirectly according to the methods described herein. There is no particular limitation as to the nature or composition of the analyte. Suitable examples of analytes include, but are not limited to, proteins, peptides, nucleic acids (including DNA and RNA), carbohydrates, lipids, steroids, and / or other small molecules. The analyte may be synthetic or non-synthetic (natural) and may come from any source. Typically an analyte will be capable of being identified, detected and / or quantified indirectly after a labelled species (e.g. a fluorescently labelled species) has first bound to the analyte, and is then examined by a suitable means like spectroscopy, fluorometry and / or microscopy.

[0168] Whilst the method of the disclosure may be used to detect binding of a labelled species to a single analyte of interest, it may also be used in multiplex applications whereby binding of multiple distinct labelled-species to their cognate analytes is detected simultaneously and / or consecutively. Thus, in some examples, the method comprises detecting one analyte of interest, or two or more analytes of interest.

[0169] As used herein, the term “labelled species” refers to any molecule that is capable of binding to an analyte of interest and which is attached directly or indirectly to a fluorophore. Exemplary labelled species include, but are not limited to, naturally-occurring and non- naturally-occurring molecules, including proteins (including antibodies), peptides, oligomers (including aptamers and nucleic acids), nucleotides, nucleosides, and small molecules. The labelled species may in some embodiments may comprise an analyte binding moiety. As used herein, “analyte binding moiety” refers to a moiety, to which the capability of the labelled species to bind to the analyte is largely attributed. There is no particular limitation as the chemical or structural form of the analyte binding moiety, for example it may be, withoutAtorney Docket No.: 67649-702601 limitation, a small molecule moiety, protein (including antibodies), peptide, oligomers (including aptamers, and nucleic acids), nucleotides, nucleosides, nucleobases, ligands, and enzymes.

[0170] The labelled species may be capable of binding to just one analyte of interest or to more than one analyte of interest. As used herein, “binding” refers to covalent-bond forming or non-covalent interaction between a labelled species and an analyte. In some embodiments, the binding is covalent. In some embodiments, the binding is non-covalent. Non-covalent binding between the labelled species may be low or high affinity. “Affinity”, “affinity of binding”, “binding affinity” and similar terms refers to the strength of a non-covalent binding interaction between a binding pair (e.g., a labelled species and an analyte of interest), and is typically represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants. Affinity can be measured by well-established methods known in the art, for example and without limitation, Surface Plasmon Resonance (SPR). In some examples, the labelled species binds specifically to the analyte of interest. Alternatively, or in addition, the labelled species binds selectively to the analyte of interest. It will be understood that in the context of the methods described herein, that descriptors as to the binding being specific and / or selective are stated relative to at least one other, or all other, analytes that are present in the method.

[0171] Depending on the specific application to which the method of the disclosure is being applied, there may be one or more than one labelled species contacted with the analyte of interest. In some embodiments, a single labelled species is contacted with the analyte of interest. In other embodiments, two or more (e.g., 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or more labelled species) are contacted with the analyte of interest. In accordance with examples in which more than one labelled species is contacted with an analyte of interest, it will be understood that each labelled species may have the same, different, or no binding selectivity vis-a-vis each analyte that is present or different component parts of an analyte.

[0172] The labelled species may be inherently fluorescent, or the labelled species may comprise a fluorophore that is attached or conjugated to a species (for example, the analyte binding moiety) via any known chemistry. Such methods are well known in the art, and there are a variety of fluorophores that are commercially available in forms that may be readily attached to molecules comprising functional groups such as primary or second amines, carboxylic acids, thiol groups, azides, alkynes or tetrazines. For such purposes, commercially available fluorophores exist in which the fluorophore comprises or is linked to a reactiveAtorney Docket No.: 67649-702601 handle such as, and without limitation, NHS-esters, maleimides, azides, alkynes (including strained alkynes such as cyclooctynes). Such commercially available reagents are typically accompanied by instructions by which the reagent may be used to attach a fluorophore to a compound having an appropriate complementary reactive functional group. Where there is more than one labelled species, each labelled species may comprise the same, or a different, fluorophore to at least one, or all, other labelled species. In some embodiments, at least labelled species comprises a fluorophore which is different to one or more other labelled species contacted with the analyte of interest. In some embodiments, each labelled species comprises a different fluorophore. In some embodiments, each labelled species comprises the same fluorophore.

[0173] The terms “fluorophore”, “fluorescent label”, or similar refer to a signalling moiety that conveys information through the fluorescent absorption and / or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer and the like. In some embodiments, the fluorophore is an organic fluorophore. As used herein “organic fluorophore” refers to all non-particulate compounds or moieties that do not contain mineral or inorganic components and are capable of fluorescence. In some embodiments, the fluorophore is an inorganic fluorophore, viz. a fluorophore that is not an organic fluorophore.

[0174] Exemplary fluorophores which may be conjugated, attached or comprised by labelled species used in the method include, but are not limited to fluorescein, Oregon Green, fluorescein isothiocyanate (FITC), 6-Carboxyfluorescein, tetramethylrhodamine, Texas Red, dansyl, Alexa Fluor 488, BODIPY FL, lucifer yellow, and Alexa Fluor 405 / Cascade Blue fluorophores.

[0175] In some embodiments, the fluorophore is selected from the group consisting of: fluorescein, rhodamine, cyanine, dipyrromethene, naphthalene, xanthene, anthracene, squaraine and triangulenium.

[0176] In some embodiments, the fluorophore is selected from the group consisting of: ATTO 565, ATTO 655, Acridine Orange, Acridine Yellow, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, BODIPY 500 / 510, BODIPY 493 / 503, BODIPY R6G, BODIPY 530 / 550, BODIPY FL, BODIPY TR-X, BODIPY TMR, BODIPY 558 / 568, BODIPYAtorney Docket No.: 67649-702601564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Coumarin 6, CY2, CY3B, CY3, CY3.5, CY5, CY5.5, CY7, Dansyl, DAPI, DPH, DYLIGHT™ DYES (e.g, DYLIGHT™ 405, DYLIGHT™ 488, DYLIGHT™ 549, DYLIGHT™ 594, DYLIGHT™ 633, DYLIGHT™ 649, DYLIGHT™ 680, DYLIGHT™ 750, DYLIGHT™ 800 and the like) (available from Thermo Fisher Scientific, Rockford, Ill.), Erythrosin, Ethidium Bromide, Fluorescein, FITC, FURA-2, GFP, Hoechst 33258, Hoechst 33342, HPTS, Indocyanine Green, KU530, KU560, Laurdan, lissamine rhodamine B, Lucifer Yellow, Marina Blue, Nile Red, Oregon Green, Oregon Green 488, Oregon Green 514, Pacific Blue, Prodan, Pyrene, rhodamine 6G, rhodamine green, rhodamine red, Rhodamine 101, Rhodamine 110, Rhodamine 123, Rhodamine 6G, Rhodamine B, Ru(bpy)3[PFe]2, Ru(bpy)2(dcpby)[PFe]2, SeTau dyes such as SeTau-380, SeTau-404, SeTau-405, SeTau-647, SeTau-425, SITS, SNART, Stilbene SITS, SITA, Texas Red, tetramethyl rhodamine, TOTO-1, YOYO-1, and YOYO-3.

[0177] In some embodiments, at least one or each of the fluorophores attached to the one or more labelled species has a fluorescence lifetime (in ns) of at least about 0.1. For example, the or each fluorophore attached to the one or more labelled species may have a fluorescence lifetime of between about 0.1 ns and about 500 ns. In one example, the or each fluorophore attached to the one or more labelled species may have a fluorescence lifetime of between about 0.1 ns and about 5ns. In one example, the or each fluorophore attached to the one or more labelled species may have a fluorescence lifetime of between about Ins and about 10 ns (e.g., such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ns). In another example, the or each fluorophore attached to the one or more labelled species has a fluorescence lifetime of between about 10 ns and about 100 ns (e.g., between about 10ns to about 30 ns, or between about 10 ns to about 50 ns, or between about 10 ns to about 70 ns).

[0178] In some embodiments, at least one or each of the fluorophores attached to the one or more labelled species has a Stokes shift (in nm) about or greater than about 5. For example, at least one or each fluorophore attached to the one or more labelled species has a Stokes shift of between about 5nm to about 30nm (e.g., such 6 nm, or 7 nm, or 8 nm, or about 9 nm, or about 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, or 29 nm or 30nm)..

[0179] Alternatively, or in addition, the fluorophore(s) of the labelled species may have a low quantum efficiency and / or yield. In this regard, compared with fluorophores which are strong emitters, poor emitters with low quantum yields may benefit from the strong fluorescentAtorney Docket No.: 67649-702601 enhancement within the area of electrical field enhancement. The use of fluorophores having low quantum yield may further aid in reducing background noise from fluorescently-labelled species outside of the area of electrical field enhancement. In some examples, at least one or each fluorophore attached to the one or more labelled species has a quantum yield of between about 1% and about 50%. For example, at least one or each fluorophore attached to the one or more labelled species may have a quantum yield of between about 1% and about 20%. For example, at least one or each fluorophore attached to the one or more labelled species may have a quantum yield of between about 10% and about 30%.

[0180] In other examples, at least one or each fluorophore attached to the one or more labelled species has a quantum yield of greater than about 50% (e.g., greater than or equal to about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0181] In the normal course of performing the method, the one or more labelled species are contacted with the analyte of interest in the presence of one or more quenchers and within an area of electrical field enhancement, and the fluorophore is irradiated. In order for fluorophores comprised by the labelled species to emit a fluorescence signal, a source of electromagnetic energy must be provided. Accordingly, the method may comprise providing a source of electromagnetic energy to the labelled species when bound to the analyte of interest. In one example, the electromagnetic energy is provided by one or more lasers. The one or more lasers will be configured to provide electromagnetic energy (e.g., light) to the labelled species at a wavelength corresponding to the excitation wavelength of the fluorophores attached to the species.

[0182] As described herein, the one or more labelled species which are contacted with the analyte of interest have distinct fluorescence emission signatures which are enhanced when the fluorophore is excited within the area of electrical field enhancement. It is the detection of the enhanced distinct fluorescence emission signature that enable a determination that a specific binding event has occurred between a labelled species (i.e., the labelled species associated with the distinct fluorescence emission signature) and the analyte of interest. The term “fluorescence emission signature”, as used herein, refers to cumulative fluorescence properties of the labelled species when excited during a process of detecting binding of a labelled species to an analyte of interest as described herein including, but not limited to, peak emission wavelength, fluorescence emission intensity, and fluorescence emission duration. Accordingly, it follows that a “distinct fluorescence emission signature” is one which can be distinguished from another fluorescence emission signature when labelled species are excited.Atorney Docket No.: 67649-702601For example, a “distinct fluorescence emission signature” as described herein may be a fluorescence emission signature for a labelled species which is distinguishable from another labelled species used in the method described herein based on differences in one or more of differences in peak emission wavelength, differences in fluorescence emission intensity, differences in fluorescence emission duration, differences in duration between successive fluorescence emissions, or any combinations thereof.

[0183] In some embodiments, at least one or each fluorophore has a peak emission wavelength (in nm) between about 350 and about 850 (e.g., between about 350 nm and about 650 nm). In some examples, the distinct fluorescence emission signatures are based on peak emission wavelength, and two or more (e.g., 2, or 3 or 4 or 5 or 6 or more) labelled species are distinguishable based on differences in peak emission wavelength. In one example, each different labelled species comprises a fluorophore having a distinct peak emission wavelength. In accordance with any example in which labelled species are distinguishable based on differences in peak emission wavelength, the peak emission wavelengths of the respective labelled species may be separated by 10 nm or more relative to each other. For example, the peak emission wavelengths may be separated by about 25 nm or more relative to each other. For example, the peak emission wavelengths may be separated by about 50 nm or more relative to each other. For example, the peak emission wavelengths may be separated by about 75 nm or more relative to each other. For example, the peak emission wavelengths may be separated by about 100 nm or more relative to each other. In certain examples, each different species is linked to a fluorophore having a distinct peak emission wavelength and the peak emission wavelengths of the respective fluorophores is separated by about 100 nm or more relative to each other.

[0184] Each labelled species may include a fluorophore having a peak emission wavelength independently selected from an emission wavelength in the visible spectrum, an emission wavelength in the ultraviolet (UV) spectrum, an emission wavelength in the infrared (IR) spectrum, and an emission wavelength in the near-IR spectrum. In one example, one or more or each fluorophore has a peak emission wavelength independently selected from an emission wavelength between about 350 nm and 850 nm (e.g., between about 350 nm and 650 nm).

[0185] Alternatively, or in addition, different species are distinguished from one another based on differences in fluorescence emission intensity. For example, differences in fluorescence intensity may be achieved by differences in the amount of a fluorophore (e.g., the same fluorophore or a different fluorophore) linked to different species. In accordanceAtorney Docket No.: 67649-702601 with this example, two or more species of labelled species used in the method may labelled with the same fluorophore and are distinguishable based on differences in fluorescence emission intensity. In one example, two or more species of labelled species used in the method are distinguishable based on differences in fluorescence emission intensity.

[0186] In another example, different labelled species are distinguished from one another based on differences in fluorescence emission duration. For example, in the context of DNA and RNA sequencing, different labelled nucleotide species are known to have different dwell times when being incorporated into the growing DNA strand by polymerase (e.g., DNA polymerase or reverse transcriptase) during synthesis of the nascent strand. These differences in dwell time impact the duration of fluorescence emission from the fluorophore linked to the labelled species. Accordingly, in some examples, the distinct fluorescence emission signature by which a species is identified during the method of the disclosure may include a fluorescence emission duration which is unique to the species, or distinct to species in combination with a specific fluorophore. In accordance with this example, two or more species of labelled species used in the method may be distinguished based on differences in fluorescence emission duration.

[0187] As described herein, the distinct fluorescence emission signatures of the respective labelled species are enhanced within the area of electrical field enhancement relative to the fluorescence emission signature(s) of corresponding labelled species outside of the area of electrical field enhancement. In one example, the distinct fluorescence emission signature of the labelled species within the area of electrical field enhancement is enhanced by two times or more (e.g., by about three times or more, or by about four times or more, or by about five times or more, or by about six times or more, or by about seven times or more, or by about eight times or more or by about nine times or more) as compared to the fluorescence emission signatures of corresponding labelled species outside of the area of electrical field enhancement. In some examples, the distinct fluorescence emission signature of the labelled species is enhanced by an order of magnitude or more (e.g., about lOx or more, or about 20x or more, or about 30x or more, or about 40x or more, or about 50x or more, or about 60x or more, or about 70x or more, or about 80x or more, or about 90x or more, or about lOOx or more) as compared to the fluorescence emission signature of corresponding labelled species outside of the area of electrical field enhancement. In further examples, the distinct fluorescence emission signature of a labelled species is enhanced by at least about lOOx or more (e.g., at least about 150x, or at least about 200x, or at least about 250x, or at least aboutAtorney Docket No.: 67649-702601300x, or at least about 350x, or at least about 400x, or at least about 450x, or at least about500x, or at least about 550x, or at least about 600x, or at least about 650x, or at least about700x, or at least about 750x, or at least about 800x, or at least about 850x, or at least about900x, or at least about 950x, or at least about lOOOx) as compared to the fluorescence emission signature of a corresponding labelled species outside of the area of electrical field enhancement.

[0188] A particular advantage of the method of the disclosure is that signal-to-noise ratio (SNR) can be improved over an analogous method in which the one or more quenchers of the fluorophore(s) are not present within the area of electrical field enhancement. The inventors postulate that this improvement in SNR may be the result of engineering the spontaneous decay rate of the fluorophore attached to the fluorescently-labelled species in two ways simultaneously. Firstly, the positioning the quencher in close proximity to the fluorophore increases the non-radiative decay rate of the fluorophore, leading to a reduction in quantum yield. Secondly, positioning of the fluorophore within an enhancement region increases the rate of radiative decay, which leads to an increase in quantum yield within the enhancement region. The inventors postulate that combining these two effects leads to reduced background noise and a recovered signal within the enhancement region, because the increase in rate of radiative decay for fluorophores within the enhancement region is relatively greater than the increase in rate of non-radiative decay for fluorophores which are quenched. Accordingly, in some embodiments, the SNR of the labelled species is increased within the area of electrical field enhancement compared to performance of the method using the corresponding labelled species without the one or more quenchers at equivalent fluorophore concentrations. The term "signal to noise ratio", “SNR”, “S / N” or similar refers to the ratio of the signal power to the noise power. As used herein, “SNR” shall be understood to mean the ratio of (‘S’), being the intensity of fluorescence from a labelled species when bound to the analyte of interest, to (‘N’), being the intensity of unwanted background fluorescence which is not associated with the binding event between the labelled species and the analyte. SNR is therefore a quantitative measurement of the quality of a signal, whereby the higher the SNR the better the quality of the signal. The person skilled in the art will appreciate that there are multiple methods for quantifying signal-to-noise, for example and without limitation, by dividing the mean signal intensity by the standard deviation of background noise. In some examples, the signal-to- noise ratio of the one or more labelled species is increased by the presence of one or more quenchers within the area of electrical field enhancement by about 5% or more (e.g., aboutAtorney Docket No.: 67649-70260110%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%, or about 150%, or about 200% or more) compared to performance of the method using the corresponding labelled species without the one or more quenchers at equivalent fluorophore concentrations. . For example, the SNR of the one or more labelled species may be increased by the presence of one or more quenchers within the area of electrical field enhancement by two times or more (e.g., by about three times or more, or by about four times or more, or by about five times or more, or by about six times or more, or by about seven times or more, or by about eight times or more or by about nine times or more) as compared to the SNR when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations. In some examples, the SNR of the one or more labelled species is increased within the area of electrical field enhancement by an order of magnitude or more (e.g., about lOx or more, or about 20x or more, or about 30x or more, or about 40x or more, or about 50x or more, or about 60x or more, or about 70x or more, or about 80x or more, or about 90x or more, or about lOOx or more) as compared to the SNR when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations.

[0189] Alternatively, or in addition, the background noise of the labelled species outside the area of electrical field enhancement may be reduced by the presence of one or more quenchers, including within the area of electrical field enhancement, compared to performance of the method using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations. As stated above, the inventors believe that this is because the increase in rate of radiative decay for fluorophores within the enhancement region (which increased quantum yield of the fluorophore) is relatively greater than the increase in rate of non-radiative decay for fluorophores which are quenched (which decreases quantum yield of the fluorophore). In some embodiments, background noise of one or more labelled species outside the area of electrical field enhancement is reduced by the presence of one or more quenchers, including within the area of electrical field enhancement, by about about 5% or more (e.g., about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%, or about 150%, or about 200% or more) compared to performance of the method using the corresponding labelled species without the one or more quenchers at equivalent fluorophore concentrations. For example, the background noise of the one or more labelled species outside the area ofAtorney Docket No.: 67649-702601 electrical field enhancement may be reduced by two times or more (e.g., by about three times or more, or by about four times or more, or by about five times or more, or by about six times or more, or by about seven times or more, or by about eight times or more or by about nine times or more) as compared to the background noise when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations. In some examples, the background noise is reduced by an order of magnitude or more as compared to the background noise when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations.

[0190] As described herein, one particular advantage of increasing the SNR and / or reducing background noise by performing the method of the disclosure is that, if desired (as it may be in certain applications), a higher concentration of fluorophore may be used for any one or more of the labelled species. This may be particularly helpful when using fluorophores with low quantum yield and / or efficiency, and / or when detecting an analyte which is present in a sample in low abundance (which may lead to fewer interactions between the analyte and labelled species and therefore fewer binding events). Total concentration of fluorophore may be increased by attaching more than one of the respective fluorophores to each labelled species, or by increasing the concentration of the labelled species in the assay. The person skilled in the art will be capable of determining the concentration of the fluorophore by any of the commonly known methods in the art, for example absorbance spectroscopy. Where the loading of a fluorophore on a labelled species is 1 : 1 (that is to say, that the labelled species comprises a single fluorophore moiety), then the concentration of that fluorophore would be expected to be equivalent to the concentration of the labelled species, accounting for whether that same fluorophore may be provided on a separate labelled species, as well as any degradation of the fluorophore moiety (e.g. photobleaching). In one example, at least one or each fluorophore attached to the one or more labelled species is present at a concentration of at least about 10 nM. For example, at least one fluorophore attached to the one or more labelled species may be present at a concentration of between about 10 nM to about 100 nM (e.g., such as about 20 nM, or about 30 nM, or about 40 nM, or about 50 nM, or about 60 nM, or about 70 nM, or about 80 nM, or about 90 nM, or about 100 nM). In certain applications, the concentration of fluorophore may exceed 100 nM e.g., about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 400 nM, or about 500 nM or more. In particularAtorney Docket No.: 67649-702601 examples, at least one or each fluorophore is present at a concentration of at least about 10 nM, or about 50 nM or about 100 nM.

[0191] As stated above, it has been surprisingly discovered that quenching of fluorescently labelled species within an area of electrical field enhancement increases the signal to noise ratio of the fluorescence emission signature associated with a binding event between that fluorescently labelled species and the analyte of interest. The presence of one or more quenchers generally, including outside the area of electrical field enhancement, may further reduce background noise, thereby improving the signal to noise ratio further. As used herein, “a quenching agent” or "quencher" refers to any fluorescence-modifying moiety that can absorb, attenuate, or reduce the light emitted by a fluorophore. This absorbance, attenuation, or reduction in emission of light is referred to as "quenching". The one or more quenchers may assist in reducing background noise from fluorophores linked to labelled species, or other fluorescent species which may be present, which are outside of the area of electrical field enhancement. In a broad sense, a quencher refers to any molecule that can reduce the amount of intensity of emissions from an fluorescence emitter. Quenchers do not have to, although they may, act by a “Forster Resonance Energy Transfer” (FRET) mechanism. A quencher may act by any possible mechanism; there does not need to be, although there may be, spectral overlap between the fluorophore and the quencher. Accordingly, in some embodiments, the fluorophore has an emission spectrum that overlaps with an absorption spectrum of the one or more quenchers. For the avoidance of doubt, quenching may occur by e.g. without limitation, dynamic quenching (Forster, Dexter, etc.), static quenching (ground state complex), energy transfer, photoelectron transfer, proton-coupled electron transfer, dimer formation between closely spaced fluorophores, transient excited state interactions, collision quenching, or formation of non-fluorescent ground state species.

[0192] Quenchers which may be useful in the methods of the disclosure include, without limitation, Black Hole Quencher Dyes (Biosearch Technologies such as BHQ-0, BHQ-1, BHQ-2, BHQ-3, BHQ-10; QSY Dye fluorescent quenchers (from Molecular Probes / Invitrogen) such as QSY7, QSY9, QSY21, QSY35, and other quenchers such as Dabeyl and Dabsyl; Cy5Q and Cy7Q and Dark Cyanine dyes (GE Healthcare), which can be used, for example, in conjunction with donors such as Cy3B, Cy3, or Cy5; DY-Quenchers (Dyomics), such as DYQ-660 and DYQ-661; and ATTO fluorescent quenchers (ATTO-TEC GmbH), such as ATTO 540Q, 580Q, 612Q. In some embodiments, the one or more quenchers are independently selected from the group consisting of: BHQ-0, BHQ-1, BHQ-2, BHQ-3,Atorney Docket No.: 67649-702601BHQ-10, QSY7, QSY9, QSY21, QSY35, Dabeyl, Dabsyl, Cy5Q, Cy7Q, DYQ-660, DYQ- 661, 540Q, 580Q, and 612Q.

[0193] The person skilled in the art will appreciate that a quencher of the fluorophore may be provided either by covalently linking the quencher to the fluorophore (i.e. within with the labelled species), separately, or a combination of both (i.e. a covalently bound quencher, and a separately provided quencher). In some embodiments, the labelled species is inherently capable of quenching the fluorophore.

[0194] Accordingly, in some embodiments, the labelled species may comprise one or more quenchers. By this it will be understood either that the labelled species is inherently capable of quenching the fluorophore, or the quencher is covalently bound to the labelled species, either directly or indirectly (e.g. via a linking moiety). In some embodiments, the labelled species comprises two or more quenchers. In some embodiments, the labelled species comprises three or more quenchers. In some embodiments, the labelled species comprises a single quencher.

[0195] In some embodiments, the labelled species comprises one or more quenchers, and the fluorophore and one or more quenchers are positioned within the labelled species such that the one or more quenchers attenuate the emission intensity of the fluorophore when the fluorophore is excited. By this it will be understood that the quencher, e.g. covalently bound to the labelled species, is always within a distance to the fluorophore, regardless of the conformation(s) that the labelled species may adopt from time to time, such that the quencher is always capable of quenching at least a portion of the emissions from the fluorophore, when the fluorophore is excited.

[0196] The one or more quenchers are positioned within the labelled species relative to the fluorophore such they are capable of quenching at least 30% (e.g., at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or at least 95% of the fluorescence emission of the fluorophore irrespective of the structural conformation of the labelled species.

[0197] The skilled person will appreciate that many quenchers are most effective at quenching when the cognate quencher and fluorophore are within a certain radius or distance. In some embodiments, the average distance between the fluorophore and the one or more quenchers within each labelled species is less than about 15nm. However, the average distance between cognate fluorophores and quenchers within a labelled species of the disclosure is preferably less than about lOnm (e.g., such as about 9 nm, or about 8 nm, or about 7 nm, or about 6 nm,Atorney Docket No.: 67649-702601 or about 5 nm, or about 4 nm, or about 3 nm, or about 2 nm, or about 1 nm, or about 0.9 nm, or about 0.8 nm, or about 0.7 nm, or about 0.6 nm, or about 0.5 nm, or about 0.4 nm, or about 0.3 nm, or about 0.2 nm). Preferably the average distance between cognate fluorophores and quenchers within a labelled species remains substantially constant, irrespective of the structural conformation of the labelled species.

[0198] Additionally, or alternatively, the quenchers may be provided in a free, unconjugated form. Thus, in some embodiments, the one or more quenchers are provided separately at a concentration that attenuates the emission intensity of the fluorophore when the fluorophore is excited. By this it will be understood that the one or more quenchers are not covalently bound, or otherwise attached, to the labelled species. For example, the one or more quenchers may be present as a separate component in a solution with the one or more labelled species and analyte of interest. Where this is the case, the one or more quenchers may be present at a concentration that is sufficient to quench at least a portion of the fluorescent emissions of the fluorophore(s) attached to the one or more labelled species. In some embodiments, the one or more quenchers are independently present at a concentration (in nM) of about or at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 100000, 1000000 or more. In some embodiments, the one or more quenchers are independently present at a concentration (in nM) of about or at least about 10, 50 or 100.Further Examples of Labelled Species

[0199] As stated above, in some embodiments the labelled species comprises the fluorophore and at least one quencher. Further examples of suitable labelled species include those disclosed in W02003087302A2 and W02000036152A1, the entire contents of which is hereby incorporated herein by reference.

[0200] In some embodiments, the labelled species is a compound of formula (I):(Formula I) wherein:R1is or comprises the fluorophore;R2is or comprises the quencher;R1and R2are optionally interchangeable;A1and A3are each independently absent or is;Atorney Docket No.: 67649-702601A2is absentL1to L3are each independently absent or a divalent linking moiety which is uninterrupted or interrupted, and optionally substituted;L4is absent or is a terminal moiety which is uninterrupted or interrupted, and optionally substituted;L5is absent or a trivalent linking moiety which is uninterrupted or interrupted, and optionally substituted; and each X is independently absent or is a analyte binding moiety, on the proviso that at least one X is present.

[0201] In some embodiments, R1is the fluorophore. R1comprises the fluorophore. In some embodiments, R2is the quencher. In some embodiments, R2comprises the quencher. In some embodiments, R1and R2are not interchangeable. It will be understood that the quencher is a quencher of the fluorophore. The fluorophore may be any fluorophore, including those described herein. Likewise, the quencher may be any quencher of the fluorophore, including those quenchers described herein.

[0202] In some embodiments, A1is absent. In some embodiments, A2is absent. In some embodiments, A3is absent. In some embodiments, A1and A3are both absent and A2is present. In some embodiments, A1and A2are both absent and A1is present. In some embodiments, A1and A2are both absent and A3is present. In some embodiments, A1is absent and A3and A2are present. In some embodiments, A2is absent and A1and A3are present. In some embodiments, A3is absent and A1and A2are present.some, embodimen+ts AA1 • I““L is *3“X“IT. .. In some embodimen+ts AA• h"^“X“L is *4.TIn someembodiments, A2is. In some embodiments, A2is X

[0204] L1to L3represent linking moieties and are each either independently absent or a divalent linking moiety which is uninterrupted or interrupted, and optionally substituted. Similarly, L4is either absent or is a terminal moiety which is uninterrupted or interrupted, and optionally substituted. Similarly, L5represents a linking moiety that is either absent or is a trivalent moiety which is uninterrupted or interrupted, and optionally substituted.Atorney Docket No.: 67649-702601

[0205] Unless otherwise stated or structurally depicted, it will be appreciated that the orientation of L1to L3, if present, within the compound of Formula (1) are undefined. That is, L1to L3, if present, may be attached at either side within the compound of Formula (1). When any one of L1to L3is absent, it will be understood that there is a direct bond between the nearest present groups on either side. Similarly, it will be appreciated that the orientation of L5if present, within the compound of Formula (1) is undefined.

[0206] In one embodiment, L1is absent or is an aliphatic linker group which is uninterrupted or interrupted and optionally substituted. In one embodiment, L2is absent or is an aliphatic linker group which is uninterrupted or interrupted and optionally substituted. In one embodiment, L3is absent or is an aliphatic linker group which is uninterrupted or interrupted and optionally substituted. In one embodiment, L4is absent or is an aliphatic linker group which is uninterrupted or interrupted and optionally substituted. In one embodiment, L5is absent or is an aliphatic linker group which is uninterrupted or interrupted and optionally substituted. In one embodiment, L1to L3are each independent absent or are aliphatic linker groups which are uninterrupted or interrupted and optionally substituted.

[0207] In one embodiment, L1is absent or is an aliphatic linker group which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, -C(=O)NR4-, -NR4- , -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and -N(R4)C(=O)N(R4)-. In one embodiment, L1is present and is -Ci-2oalkyl-, which is uninterrupted or interrupted and optionally substituted. In one embodiment, L1is -Ci-ioalkyl- which is uninterrupted or interrupted and optionally substituted. In one embodiment, L2is absent or is an aliphatic linker group which is uninterrupted or interrupted with one or more groups selected from -O- , -S-, -C(=O)-, -C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and -N(R4)C(=O)N(R4)-. In one embodiment, L2is present and is -Ci-2oalkyl-, which is uninterrupted or interrupted and optionally substituted. In one embodiment, L2is -Ci-ioalkyl- which is uninterrupted or interrupted and optionally substituted.

[0208] In one embodiment, L3is absent or is an aliphatic linker group which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, -C(=O)NR4-, -NR4- , -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and -N(R4)C(=O)N(R4)-. In one embodiment, L3is present and is -Ci-2oalkyl-, which is uninterrupted or interrupted and optionally substituted. In one embodiment, L3is -Cl-lOalkyl- which is uninterrupted or interrupted and optionally substituted.Atorney Docket No.: 67649-702601

[0209] In one embodiment, L4is absent or is an aliphatic terminal moiety which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, - C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and - N(R4)C(=O)N(R4)-. In one embodiment, L4is present and is -Ci-2oalkyl-, which is uninterrupted or interrupted and optionally substituted. In one embodiment, L4is -Ci-ioalkyl- which is uninterrupted or interrupted and optionally substituted.

[0210] In one embodiment, L5is absent or is an aliphatic trivalent moiety which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, - C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and - N(R4)C(=O)N(R4)-. In one embodiment, L5is present and is -Ci-2oalkyl-, which is uninterrupted or interrupted and optionally substituted. In one embodiment, L5is -Ci-ioalkyl- which is uninterrupted or interrupted and optionally substituted.

[0211] In one embodiment, L1to L5may be each optionally substituted. In one embodiment, each of L1to L5where present is optionally substituted with one or more R5. In one embodiment, each of L1to L5where present is -Ci-ioalkyl- or -C2-ioalkyl- and is optionally substituted with one or more R5. In one embodiment, each of L1to L5where present is -C2- walkyl- and is optionally substituted with one or more R5. In one embodiment, each of L1to L5where present is -Ci-ioalkyl- and optionally substituted with one or more R5. In an instance of L1to L5not being substituted with one or more R5, then it will be understood that a hydrogen atom will remain as the substitution.

[0212] In one embodiment, each L1to L5where present is optionally substituted with one or more groups selected from Ci-ioalkyl, OCi-ioalkyl, -NH2, -OH, -COOH, and -C(=O)OCi- ealkyl.

[0213] In one embodiment, each of L1to L5where present, is -Cl-20alkyl- or -Cl-10alkyl-, substituted with one or more R5. In one embodiment, each of L1to L5where present is -Ci- 2oalkyl- or -Ci-ioalkyl-, substituted with one or more groups selected from Ci-ioalkyl, OCi- walkyl, -NH2, -OH, -COOH, and -C(=O)OCi-6alkyl. In one embodiment, each of L1to L5where present is -Ci-2oalkyl- or -Ci-ioalkyl-, substituted with one or more groups selected from Ci-ioalkyl, OCi-ioalkyl, -NH2, -OH, -COOH, and -C(=O)OCi-6alkyl. In one embodiment, each of L1to L5where present is -Ci-2oalkyl- or -Ci-ioalkyl-, substituted with one or more groups selected from Ci-ioalkyl, OCi-ioalkyl, -NH2, -OH, -COOH, and -C(=O)OCi-6alkyl.

[0214] In one embodiment, each of L1to L5where present is -Ci-ioalkyl-, substituted with one or more R8. In one embodiment, each of L1to L5where present is -Ci-ioalkyl-, substitutedAtorney Docket No.: 67649-702601 with one or more groups selected from Ci-ioalkyl, OCi-ioalkyl, -NH2, -OH, -COOH, and - C(=O)OCi-6alkyl. In one embodiment, each of L1to L5where present is -Cl-10alkyl-, substituted with one or more groups selected from Ci-ioalkyl, OCi-ioalkyl, -NH2, -OH, - COOH, and -C(=O)OCi-6alkyl. In one embodiment, each of L1to L5where present is or -Ci- walkyl-, substituted with one or more groups selected from Ci-ioalkyl, OCi-ioalkyl, -NH2, - OH, -COOH, and -C(=O)OCi-6alkyl.

[0215] In some embodiments, L1to L5are each independently absent or an aliphatic group which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)- , -C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and - N(R4)C(=O)N(R4)-, and is optionally substituted with one or more R5.

[0216] In some embodiments, L1to L5are each independently absent or is Ci-3oalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, - C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and - N(R4)C(=O)N(R4)-, and is optionally substituted with one or more R5. In some embodiments, L1to L5are each independently absent or is Ci-2oalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, -C(=O)NH-, -C(=O)O-. In some embodiments, L1to L5are each independently absent or is Ci-isalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, -C(=O)NH-, -C(=O)O- . In some embodiments, L1to L5are each independently absent or is Ci-ioalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, - C(=O)NH-, -C(=O)O-.

[0217] For each of the embodiments described herein, R4is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, alkylcarbocyclyl, and alkylheterocyclyl, each of which is optionally substituted with one or more R5.

[0218] For each of the embodiments described herein, R5is independently selected from the group consisting of H, halogen, Ci-ioalkyl, OCi-ioalkyl, Ci-iohaloalkyl, OCi-iohaloalkyl, C2- walkenyl, C2-ioalkynyl, OC2-ioalkenyl, OC2-ioalkynyl, 3-10 membered carbocyclyl, 3-10- membered heterocyclyl, Ci-ioalkyl-3-10-membered-carbocyclyl, Ci-ioalkyl-3-lO-membered- heterocyclyl, -NO2, -CN, -SCN, -N3, =0, -N(R5)2, -C(=O)N(R5)2, -S(=O)N(R5)2, - S(=O)2N(R5)2, -OR5, -SR5, OC(=O)R5, -C(=O)R5, -C(=O)OR5-S(=O)R5, -S(=O)2R5, - S(=O)OR5, -S(=O)2OR5, -S(=O)(OR5)2, -OS(=O)R5, -OS(=O)2R5, -OS(=O)OR9, - OS(=O)2OR5, -OS(=O)(OR5)2, -N(R5)C(=O)R5, -N(R5)S(=O)R5, N(R5)C(=O)N(R5)2, - N(R5)S(=O)2R5, -P(=O)(OR5)2, -P(=O)OR5(R5), -P(=O)(R5)2, -OP(=O)(OR5)2, -Atorney Docket No.: 67649-702601OP(=O)OR5(R5) and -OP(=O)(R5)2, wherein each Ci-ioalkyl, Ci-iohaloalkyl, C2-ioalkenyl, C2- walkynyl, 3-10 membered-carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R6.

[0219] For each of the embodiments described herein, R6is independently selected from the group consisting of H, Ci-ealkyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, Ci-6alkyl-3-10-membered-carbocyclyl, and Ci-6alkyl-3-10-membered-heterocyclyl; wherein each Ci-ealkyl, 3-10-membered-carbocyclyl, and 3-10-membered heterocyclyl is optionally substituted with one or more R7.

[0220] For each of the embodiments described herein, R7is independently selected from the group consisting of H, halogen, -NO2, -N(R8)2, -CN, -SCN, -N3, =O, -C(=O)R8, -C(=O)OR8, -C(=O)N(R8)2, -N(R8)C(=O)R8, -OR8, -P(=O)(OR8)2, -P(=O)OR8(R8), -P(=O)(R8)2, Ci-6alkyl, and -OCi-ealkyl.

[0221] For each of the embodiments described herein, R8is independently selected from the group consisting of H, Ci-ioalkyl, Ci-ioalkyl, C2-ioalkenyl, C2-ioalkynyl, 3-10 membered carbocyclyl, 3-10 membered heterocyclyl, Ci-ioalkyl-3-lO-membered carbocyclyl, Ci-ioalkyl- 3- 10-membered heterocyclyl.

[0222] It will be understood that the labelled species must comprise at least one X, wherein X is an analyte binding moiety, to which the capability of the labelled species to bind to the analyte is largely attributed. In some embodiments, X is selected from the group consisting of: small molecule moiety, protein (including antibodies), peptide, oligomers (including aptamers, and nucleic acids), nucleotides, nucleosides, nucleobases, ligands, and enzymes. In some embodiments, X is a small molecule moiety. In some embodiments, X is an antibody or fragment or derivative thereof. In some embodiments, X is an aptamer or fragment or derivative thereof. In some embodiments, X is nucleic acid or derivative thereof. In some embodiments, X is a nucleotide.

[0223] The labelled species according to Formula (I) may comprise more than one X. In some embodiments, one of A1to A3comprise an X that is an analyte binding moiety. In some embodiments, two of A1to A3comprise an X that is an analyte binding moiety. In some embodiments, each of A1to A3comprise an X that is an analyte binding moiety. In some embodiments, A1and A2each comprise an X that is an analyte binding moiety. In some embodiments, A1and A3each comprise an X that is an analyte binding moiety. In some embodiments, A2and A3each comprise an X that is an analyte binding moiety. In some embodiments, A1comprises an X that is an analyte binding moiety. In some embodiments,Atorney Docket No.: 67649-702601A2comprises an X that is an analyte binding moiety. In some embodiments, A3comprises an X that is an analyte binding moiety.

[0224] In some embodiments, X is a nucleic acid. In some embodiments, X is a nucleic acid sequence comprising and / or consisting of 1 to 100, 1 to 80, 1 to 60, 1 to 40, 1 to 20 or 1 to 10 nucleotides. In some embodiments, X is a nucleic acid sequence comprising and / or consisting of 1 to 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2 nucleotides. In some embodiments, X comprises and / or consists of a single nucleotide. In some embodiments, X comprises and / or consists of a single nucleotide selected from the group consisting of: adenine nucleotide (A), a guanosine nucleotide (G), a thymine nucleotide (T), or a cytosine nucleotide (C).

[0225] In some embodiments, X is a polypeptide. In some embodiments, X is an antigenbinding polypeptide. In some embodiments, X is an antibody or fragment thereof. In some embodiments, X is an antigen or fragment thereof.

[0226] In some embodiments, the labelled species is a compound of formula (I) that is a compound of any one of formulae (II) to (IV):R1~~L1~*X~L2~~R2Formula (II)Formula (III)Formula (IV) wherein: R1to R8, L1to L5and X are defined according to any combination of the embodiments disclosed in respect of each of R1to R8, L1to L5and X described herein.Area of Electrical Field Enhancement

[0227] As the skilled person would appreciate, there is no particular limitation as to the means by which the area of electrical field enhancement is provided or produced in the method of the disclosure. As used herein, the phrase “area of electrical field enhancement” refers to an area or region in which the electrical field is enhanced, relative to the electrical field outsideAtorney Docket No.: 67649-702601 the defined area. The person skilled in the art will appreciate that there are variety of means by which an area of electrical field enhancement may be provided. An area of electrical field enhancement may be provided by, for example and without limitation, surface structures (including nanostructured surfaces), confinements such as waveguides (including zero-mode waveguides) and microcavities, nanoparticles (including single nanoparticles or multiple nanoparticles, including multiple nanoparticle structures (such including nanoantennae), other metal structures, and combinations thereof. The person skilled in the art will appreciate that nanostructures or nanoparticles that provide an area of electrical field enhancement may be described as plasmonic.

[0228] The person skilled in the art will appreciate that the method may be performed with an area of electrical field enhancement provided by any means, provided that it permits irradiation of the analyte-bound labelled species within the area of electrical field enhancement. Typically, although not necessarily, this will involve positioning the analyte of interest, at least temporarily, within the area of the electrical field enhancement in order to facilitate the enhancement of the distinct fluorescence emission signature of the analyte-bound labelled species. This may be done, e.g., by tethering, anchoring, or immobilizing the analyte within the area of electrical field enhancement (e.g. the plasmonic hotspot). Such tethering may be accomplished by any suitable means known in the art, for example a covalent bond may be formed between the analyte and a surface, material, or compound, optionally the surface, material, or compound that is providing the area of electrical field enhancement. Suitable means for chemical tethering include, but are not limited to, tethering via a thiol, silane, amine, azide, alkyne or carboxylic acid group, or other forms of click chemistry. Molecular linkers or spacers may be used to control the distance between the analyte that the attachment surface in order to optimize interaction with the area of the electrical enhancement. Alternatively, non-covalent forms of tethering may be used, for example and without limitation, antibody-antigen interactions, aptamer interactions, protein-protein interactions, host-guest chemistry, or hybridization (e.g. nucleic acid hybridization). In one example, a DNA origami structure is used to position analytes within the area of electrical field enhancement, optionally the plasmonic hotspot. In one example, the analyte is tethered directly or indirectly via nucleic hybridization.

[0229] In some embodiments, the area of electrical field enhancement is provided by a plasmonic nanostructure, such as a plasmonic nanostructured surface, or one or more plasmonic nanoparticles. As used herein “plasmonic nanostructure” refers to any independentAtorney Docket No.: 67649-702601 nanostructure that exhibits surface plasmon resonance, localized surface plasmon resonance, or coupled plasmon resonance properties. Plasmonic structures are well known in the art, for example, plasmonic nanostructured surfaces and plasmonic nanoantennae and methods for their construction are known in the art, see for example A. Minopoli et al., ‘Nanostructured Surfaces as Plasmonic Biosensors: A Review’, Adv. Mater. Interfaces, 2022, 9, 2101133 and Giannini et al., Plasmonic Nanoantennas: Fundamentals and Their Use in Controlling the Radiative Properties of Nanoemitters’ Chemical Reviews, 2011, 111(6), 3888-3912, the entire contents of each of which is incorporated herein by reference.

[0230] In some embodiments, the area of electrical field enhancement is provided by a plasmonic nanostructured surface. In some embodiments, the area of electrical field enhancement is provided by optical confinement, such as waveguides including zero mode waveguides. In some embodiments, the area of electrical field enhancement is provided by zero-mode waveguides. Zero mode waveguides (ZMWs) are generally characterized by the existence of a core surrounded by a cladding, where the core is dimensioned such that it precludes a substantial amount of electromagnetic radiation that is above a cut-off frequency from propagating through the core. As a result, when illuminated with light of a frequency below the cutoff frequency, the light will only penetrate a short distance into the core, effectively illuminating only a small fraction of the core's volume. ZMWs, their fabrication, structure, and use in analytical operations are described in detail in U.S. Pat. No. 6,917,726 and Levene, et al., Science 299(5607):609-764 (2003), the entire contents of which is hereby incorporated herein by reference.

[0231] In some embodiments, the area of electrical field enhancement is provided by one or more plasmonic nanoparticles. In some embodiments, the area of electrical field enhancement is provided by a nanostructure comprising two or more plasmonic nanoparticles. In some embodiments, the area of electrical field enhancement is provided by a plasmonic nanoantennae e.g., as described herein.

[0232] In some embodiments, the area of electrical field enhancement is provided by a plasmonic hotspot. The terms “plasmonic hotspot", “plasmon enhancement hotspot” or similar are used herein to define an area or region between two or more plasmonic nanoparticles where the electrical field is enhanced relative to an electrical field outside the area or region defined between the two or more plasmonic nanoparticles. In one example, a “plasmonic hotspot” is an area of electrical field enhancement due to plasmon resonance i.e., polarization of the plasmonic nanoparticles, induced by the incoming oscillating electric fieldAtorney Docket No.: 67649-702601(e.g. light). The person skilled in the art will appreciate that plasmonic hotspots may be produced by a variety of means, including but not limited to plasmonic nanoantenna. In some embodiments, the plasmonic hotspot is produced by a plasmonic nanostructure. In some embodiments, the plasmonic hotspot is produced by a plasmonic nanoantenna. Suitable plasmonic nanoantenna include, but are not limited to, those described herein.

[0233] As used herein, the term “plasmonic nanoparticle” refers to a particle having nanosized dimensions whose electron density can couple with electromagnetic radiation of wavelengths that are far larger than the particle due to the nature of the dielectric-metal interface between the medium and the particles. It will be appreciated that plasmonic nanoparticles are capable of exhibit differing scattering, absorbance, and coupling properties based on their geometries and relative positions, and different geometries are contemplated herein. The nanoparticles may be formed of any material which possesses plasmonic resonance properties. Suitable nanoparticles include, but are not limited to, metallic nanoparticles, metal alloy nanoparticles, polymeric nanoparticles and derivatives and composites thereof. In one particular example, each of the plasmonic nanoparticles are independently selected from metallic nanoparticles.

[0234] As used herein, the term “metallic nanoparticle” shall be understood to mean a particle having nanosized dimensions and formed of a metal substance. It will be appreciated the nanoparticles may be formed in any suitable or desired shape and size, examples of which are described herein. The metallic nanoparticles may be formed wholly, or in part, of any metallic material which possesses plasmonic resonance properties. The metallic nanoparticles of the plasmonic nanoantenna described herein may be formed, either wholly or in part, of any metallic material known in the art which possesses plasmonic resonance properties. Metallic materials known to possess plasmonic resonance properties include noble metals and nonnoble metals. Suitable examples include, but are not limited to, gold, silver, aluminium, copper, bismuth, nickel, palladium and platinum nanoparticles or an alloy of any one thereof. For example, one or both or each of the metallic nanoparticles may be a gold nanoparticle, a gold alloy nanoparticle or a nanoparticle having a gold or gold alloy coating. For example, one or both or each of the nanoparticles may be a silver nanoparticle, a silver alloy nanoparticle, or a nanoparticle having a silver or silver alloy coating. For example, one or both or each of the nanoparticles may be an aluminium nanoparticle, an aluminium alloy nanoparticle or a nanoparticle having an aluminium or aluminium alloy coating. For example, one or both or each of the nanoparticles may be a copper nanoparticle, a copper alloy nanoparticle or a nanoparticle having a copper or copper alloy coating. For example, one orAtorney Docket No.: 67649-702601 both or each of the nanoparticles may be a bismuth nanoparticle, a bismuth alloy nanoparticle or a nanoparticle having a bismuth or bismuth alloy coating. For example, one or both or each of the nanoparticles may be a nickel nanoparticle, a nickel alloy nanoparticle or a nanoparticle having a nickel or nickel alloy coating. For example, one or both or each of the nanoparticles may be a palladium nanoparticle, a palladium alloy nanoparticle, or a nanoparticle having a palladium or palladium alloy coating. For example, one or both or each of the nanoparticles may be a platinum nanoparticle, a platinum alloy nanoparticle, or a nanoparticle having a platinum or platinum alloy coating.

[0235] In accordance with examples in which the plasmonic nanoparticles comprise a core which is coated in a metallic substance which possesses plasmonic resonance properties as described herein, the core may be formed of glass, polymer or a composite core or other suitable material.

[0236] Nanoparticles formed of non-metallic materials and metal-oxides are also contemplated for use as plasmonic nanoparticles. For example, one or more or each of the plasmonic nanoparticles may be formed of a non-metallic material or a metal oxide doped to possess or enhance plasmon resonance properties. Exemplary non-metal materials include, but are not limited to, metal oxides, chalcogenides, phosphides, nitrides and silicon. Doped plasmonic nanoparticles and methods of producing same are described in the literature and are known to the skilled person, including, but not limited to, photodoping, chemical doping, hierarchical doping and combinations thereof.

[0237] Accordingly, in one example, one or more of the plasmonic nanoparticles is formed of non-metallic material or metal oxide which has been photodoped. Methods of photodoping nanoparticles to impart or enhance plasmon resonance properties which may be employed herein are described in Petrini et al., (2023) J. Phys. Chem. C. Nanomater Interfaces, 127(3): 1576-1587. In another example, one or more of the plasmonic nanoparticles is formed of non-metallic material or a metal oxide which has been doped hierarchically. Methods of hierarchically doping nanoparticles to impart or enhance plasmon resonance properties which may be employed herein are described in Kim et al., (2023) Nano Letters, 23(16):7633-7641 and Russo et al. (2018) Materials & Designs, 156:311-319. In another example, one or more of the plasmonic nanoparticles is formed of non-metallic material or metal oxide which has been chemically doped e.g., by introducing metal atoms into host lattice or structure. Examples of materials which may be chemically doped to increase the carrier density and plasmon resonance properties including metal oxides, chalcogenides, phosphides, nitrides andAtorney Docket No.: 67649-702601 silicon. Methods of chemically doping nanoparticles to impart or enhance plasmon resonance properties which may be employed herein are described in Chowdhury et al., (2017) Nanoscale, 9: 15591-15597 and Liu et aL, (2019) Nature Communications, 10: 1394.

[0238] In some examples, the plasmonic nanoparticles are formed of or comprise the same material. For example, the plasmonic nanoparticles may each be metallic plasmonic nanoparticles as described herein. For example, the plasmonic nanoparticles may each be gold nanoparticles or nanoparticles which are coated in gold such that they have a gold surface. For example, the plasmonic nanoparticles may each be silver nanoparticles or nanoparticles which are coated in silver such that they have a silver surface.

[0239] In other examples, the plasmonic nanoparticles are formed of or comprise different materials to one another. For example, one nanoparticle may be a gold nanoparticle or a nanoparticle coated in gold, and one or more other nanoparticles may be formed of or coated in another material (e.g., another noble metal (such as silver) or other metal with plasmonic resonance properties as described herein).

[0240] The plasmonic nanoparticles described herein may be formed in any suitable or desired shape and size, for example, as a nanosphere, nanorod, nanoprism, nanocube, nanoshell, nanotube, or nanostar. It is to be understood that these are only representative examples of nanoparticles shapes, and are in no way limiting on the disclosure. In one example, one or more or each of the plasmonic nanoparticles are nanospheres. In one example, one or more or each of the plasmonic nanoparticles are nanorods. In one example, one or more or each of the plasmonic nanoparticles are nanoprisms. In one example, one or more or each of the plasmonic nanoparticles are nanocubes. In one example, one or more or each of the plasmonic nanoparticles are nanoshells. In one example, one or more or each of the plasmonic nanoparticles are nanotubes. In one example, one or more or each of the plasmonic nanoparticles are nanostars. The plasmonic nanoparticle on the plasmonic nanoantenna may be the same shape or they may be heterogeneous.

[0241] In addition to shape, a skilled person will appreciate that the size of the nanoparticles may vary. In some examples, the plasmonic nanoparticles may have the same, or substantially the same, largest diameter. For example, each of the plasmonic nanoparticles may have a largest diameter in the range of about 5 nm to about 500 nm (e.g., in the range of about 10 nm to about 250 nm, or in the range of about 20 nm to about 200 nm, or in the range of about 50 nm to about 150 nm, or in the range of about 80 nm to about 120 nm). In some examples, each of the plasmonic nanoparticles has a largest diameter of about 80 nm. In some examples,Atorney Docket No.: 67649-702601 each of the plasm onic nanoparticles has a largest diameter of about 100 nm. In some examples, each of the plasmonic nanoparticles has a largest diameter of about 120 nm.

[0242] Where a nanostructure comprises multiple nanoparticles (e.g. in a nanoantenna), the largest diameter may differ between plasmonic nanoparticles. In accordance with an example in which the largest diameter differs between plasmonic nanoparticles, the plasmonic nanoparticles may have an average largest diameter in the range of about 5 nm to about 500 nm. For example, the plasmonic nanoparticles may have an average largest diameter in the range of about 10 nm to about 250 nm (e.g., . in the range of about 20nm to about 200nm, or about 30 nm to about 200 nm, or about 40 nm to about 200 nm, or about 50 nm to about 200 nm, or about 60 nm to about 200 nm, or about 70 nm to about 200 nm, or about 80 nm to about 200 nm, or about 90 nm to about 200 nm, or about 100 nm to about 200 nm, or about 50 nm to about 150 nm, or about 75 nm to about 150 nm, or about 100 nm to about 120 nm, or about 100 nm to about 150 nm) In one example, the average diameter of the nanoparticles is about 100 nm. In one particular example, the plasmonic nanoparticles are gold or gold- coated nanoparticles (e.g., nanospheres) having an average diameter of about 100 nm. In another example, the plasmonic nanoparticles are silver or silver-coated nanoparticles (e.g., nanospheres) having an average diameter of about 100 nm.Detection of enhanced distinct fluorescence emission signatures

[0243] In each of the foregoing examples, step (II) of the method of comprises detecting the enhanced distinct fluorescence emission signature of a labelled species that is within the area of electrical field enhancement. Such a detection thereby indicates that the labelled species is bound to the analyte of interest. This is because the labelled species, which binds to the analyte of interest, has been contacted with the analyte of interest in the area of electrical field enhancement.

[0244] The enhanced distinct fluorescence emission signatures may be detected by any means known in the art for detecting fluorescence, including but not limited to, confocal microscopy, confocal laser scanning microscopy, Total Internal Reflection (TIR), Total Internal Reflection Fluorescence (TIRF), epifluorescence microscopy, near-field scanning microscopy, far-field confocal microscopy, wide-field epi-illumination, light scattering, dark field microscopy, photoconversion, wide field fluorescence, single and / or multi-photon excitation, spectral wavelength discrimination, evanescent wave illumination, scanning two-photon, scanning wide field two-photon, Nipkow spinning disc, and / or multi-foci multi-photon. In some examples, a combination of these methods is used for detecting fluorescence. In one example,Atorney Docket No.: 67649-702601 confocal laser scanning microscopy is used to detect the enhanced distinct fluorescence emission signatures of labelled species as it binds to the analyte within the area of electrical field enhancement. In one example, Total Internal Reflection (TIR) is used to detect the enhanced distinct fluorescence emission signatures of the labelled species as it binds to the analyte within the area of electrical field enhancement. In one example, Total Internal Reflection Fluorescence (TIRF) is used to detect the enhanced distinct fluorescence emission signatures of labelled species as its binds to the analyte within the area of electrical field enhancement.

[0245] The enhanced distinct fluorescence emission signatures may be also be detected by use of any analytic device, such as “optode” element such as that disclosed in US61 / 306,235, the entire contents of which are incorporated herein for all purposes by this reference. Furthermore, such analytical devices may be arranged in an array, such as a photodiode or sensor array (which may optionally be fabricated on a silicon chip), such as those disclosed in US9488584B2 or US11415745B2, the entire contents of which are incorporated herein for all purposes by this reference.

[0246] The distinct fluorescence emission signatures emitted from the one or more labelled species may be resolved using any suitable discrimination methods which are based on: fluorescence resonance energy transfer measurements; photoconversion; fluorescent lifetime measurements; polarization; fluorescent lifetime determination; correlation / anti-correlation analysis; Raman; intensity; ratiometric; time-resolved methods; anisotropy; near-field or far field microscopy; fluorescence recovery after photobleaching (1-RAP); spectral wavelength discrimination; measurement and separation of fluorescence lifetimes; fluorophore identification; background suppression, parallel multi-color imaging, or any combination thereof. See, for example, J. R. Lakowitz 2006, in: “Principles of Fluorescence Spectroscopy”, Third Edition. If the different species are labeled with different energy transfer or reporter moieties, then resolving the emitted signals can be used to distinguish between the different species as they bind to an analyte. In one particular example, each of the labelled species emits a fluorescence signal in a different wavelength, such that the distinct fluorescence emission signatures of the labelled species are discriminated based on emission wavelength e.g., peak emission wavelength.

[0247] In order to detect distinct fluorescence emission signatures emitted from each of the different species of labelled species, the method may further employ a multifluorescence imaging system which is capable of detecting the multiple distinct fluorescence emissionAtorney Docket No.: 67649-702601 signatures emitted by the different species of labelled nucleotides during the course of the sequencing reaction. Such a system can include special filter combinations for each excitation line and / or each emission band. In one example, the detection system includes tunable excitation and / or tunable emission fluorescence imaging.

[0248] Where the method of the disclosure is performed on an array e.g., an array of plasmonic nanoantenna, the detection system may comprise an optical train which directs signals emitted from an organized array onto different locations of an array -based detector to detect multiple optical signals from multiple locations. The optical trains typically include an arrangement of optical components (including, but not limited to, lenses, mirrors, optical gratings e.g., diffraction gratings, and / or prisms e.g., wedge prisms), to simultaneously direct and separate signals having differing spectral characteristics from different addressable locations in an array to different locations on an array-based detector, e.g., a CCD.

[0249] Following the detection of the enhanced distinct fluorescence emission signature in step (II), it is indicated that the labelled species is bound to the analyte of interest.

[0250] It will be appreciated that in some embodiments, steps (I) and (II) either separately or together, may be repeated and / or performed distinctly, optionally concurrently, for the purposes of detecting binding of another labelled species to one or more analytes of interest that are present in the same solution. By this it will be understood that where multiple labelled species and analytes are present in a solution, steps (I) and (II) may be performed repeatedly, and distinctly in respect of each labelled species, as time progresses, in order to detect multiple instances of a binding between a labelled species and analyte, and / or the bindings of different labelled species to different analytes. In such a case, it will be understood by the person skilled in the art, that the method is broadly applicable to a variety of methods, for example, and without limitation, methods of sequencing a nucleic acid molecule or a polypeptide.Methods of sequencing a nucleic acid molecule

[0251] As described hereinabove, the method of detecting binding of a labelled species to an analyte of interest may be a method of sequencing a nucleic acid molecule. In some embodiments, the method is a method of sequencing a nucleic acid molecule. It will therefore be understood that any of the embodiments described in the context of the method of detecting binding of a labelled species to an analyte of interest are equally applicable mutatis mutandis to the method of sequencing a nucleic acid molecule described herein. Accordingly, in some examples, the method of detecting binding of a labelled species to an analyte of interest as descried herein is a method of sequencing a nucleic acid molecule, wherein:Atorney Docket No.: 67649-702601 the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a mixture of labelled nucleotides for a time and under conditions such that one of the labelled nucleotides binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, wherein the complementary nucleotide is positioned within an active site of the nucleic acid polymerase; wherein:(i) the mixture of labelled nucleotides comprises a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore and one or more quenchers are each linked directly or indirectly to the respective nucleotides, and are positioned relative to one another on the nucleotides such that the one or more quenchers attenuate emission intensity of the fluorophore when the fluorophore is excited,(iii) each of the labelled nucleotides A, G, T and C has a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited,(iv) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(v) the distinct fluorescence emission signatures of the labelled nucleotides are enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelled nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature; wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emissionAtorney Docket No.: 67649-702601 signatures detected as the labelled nucleotides sequentially bind to complementary nucleotides within the template polynucleotide strand and form ternary complexes with the complementary nucleotides and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand.

[0252] Accordingly, in one example, the method of sequencing a nucleic acid analyte comprises:(I) contacting the nucleic acid polymerase with the nucleic acid analyte and a mixture of labelled nucleotides, wherein the nucleic acid analyte is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand, wherein the contacting is for a time and under conditions such that one of the labelled nucleotides in the mixture binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, wherein the complementary nucleotide is positioned within an active site of the nucleic acid polymerase, and wherein:(i) the mixture of labelled nucleotides comprises a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore and one or more quenchers are each linked directly or indirectly to the respective nucleotides, and are positioned relative to one another on the nucleotides such that the one or more quenchers attenuate emission intensity of the fluorophore when the fluorophore is excited,(iii) each of the labelled nucleotides A, G, T and C has a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited,(iv) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(v) the distinct fluorescence emission signatures of the labelled nucleotides are enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(II) detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelledAtorney Docket No.: 67649-702601 nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature; wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the labelled nucleotides sequentially bind to complementary nucleotides within the template polynucleotide strand and form ternary complexes with the complementary nucleotides and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand.

[0253] In other examples, the method of detecting binding of a labelled species to an analyte of interest as descried herein is a method of sequencing a nucleic acid molecule, wherein: the or each labelled species is a labelled nucleotide; the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a mixture of labelled nucleotides in the presence of one or more quenchers for a time and under conditions such that one of the labelled nucleotides binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, wherein the complementary nucleotide is positioned within an active site of the nucleic acid polymerase; each of the following are satisfied:(i) the mixture of labelled nucleotides comprises a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore is linked directly or indirectly to each of the nucleotides, wherein each fluorophore has an emission spectrum when excited which overlaps with an absorption spectrum of one or more of the quenchers,(iii) the one or more quenchers are not attached to the labelled nucleotides and are provided at a concentration that attenuates fluorescence emission intensity of the fluorophores when the fluorophores are excited,Atorney Docket No.: 67649-702601(iv) each of the labelled nucleotides A, G, T and C has a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited,(iv) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(v) the distinct fluorescence emission signatures of the labelled nucleotides are enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelled nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature; wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the labelled nucleotides sequentially bind to complementary nucleotides within the template polynucleotide strand, and form ternary complexes with the complementary nucleotides and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand.

[0254] Accordingly, in one example, the method of sequencing a nucleic acid analyte comprises:(I) contacting the nucleic acid polymerase with the nucleic acid analyte, a mixture of labelled nucleotides, and one or more quenchers, wherein the nucleic acid analyte is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand, wherein the contacting is for a time and under conditions such that one of the labelled nucleotides in the mixture binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, wherein the complementary nucleotide is positioned within an active site of the nucleic acid polymerase; wherein:Atorney Docket No.: 67649-702601(i) the mixture of labelled nucleotides comprises a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore is linked directly or indirectly to each of the nucleotides, wherein each fluorophore has an emission spectrum when excited which overlaps with an absorption spectrum of one or more of the quenchers,(iii) the one or more quenchers are not attached to the labelled nucleotides and are provided at a concentration that attenuates fluorescence emission intensity of the fluorophores when the fluorophores are excited,(iv) each of the labelled nucleotides A, G, T and C has a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited,(iv) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(v) the distinct fluorescence emission signatures of the labelled nucleotides are enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(II) detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelled nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature; wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the labelled nucleotides sequentially bind to complementary nucleotides within the template polynucleotide strand, and form ternary complexes with the complementary nucleotides and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand.

[0255] The method of sequencing a nucleic acid molecule as described herein may be a method of sequencing-by-synthesis (SBS, also referred to as ‘sequencing by incorporation’) or a method of sequencing-by-binding (SBB). Common SBS methods employ the use a polymerase enzyme (e.g., DNA polymerase or RNA transcriptase) to synthesize a nascent DNA strand which is complementary to a template strand of DNA or RNA (as appropriate)Atorney Docket No.: 67649-702601 to be sequenced, by providing labelled nucleotides so that the base type of the incorporated nucleotide is detected as synthesis proceeds. Detection may be in real-time wherein the nucleotides are detected as they bind to a cognate (or complementary) nucleotide within the active site of the polymerase and are then incorporated into the nascent strand. Detection may also proceed in iterations of stop and proceed steps, wherein controlled reaction conditions and / or reagents reversibly stop and start the reaction at a given time during synthesis. Sequencing-by-binding (SBB), on the other hand, is generally based on repetitive cycles of (i) detecting a stabilized complex that forms at each position along the template (e.g. a ternary complex that includes the primed template, a polymerase, and a cognate nucleotide for the position which is detectably labelled) under conditions that prevent covalent incorporation of the cognate nucleotide into the primer, and (ii) extending the primer under conditions which selectively incorporate an unlabeled equivalent of the cognate nucleotide having a label to allow detection of the next position along the template. In SBB, detection of the nucleotide at each position of the template occurs prior to extension of the primer to the next position.

[0256] The term “ternary complex” is used herein in the context of both SBS and SBB to refer to the intermolecular association between a nucleic acid polymerase, a duplex region formed between a primer and template polynucleotide strand (‘primer / template duplex’), and a nucleotide. Typically, the polymerase facilitates interaction between a next correct nucleotide and a template polynucleotide strand of the primed nucleic acid analyte. A next correct nucleotide can interact with the template strand via Watson-Crick hydrogen bonding. In some examples, the ternary complex is a stabilized ternary complex. The term “stabilized ternary complex” means a ternary complex having promoted or prolonged existence, or a ternary complex for which disruption has been inhibited. Generally, stabilization of the ternary complex prevents covalent incorporation of the nucleotide component of the ternary complex into the primed nucleic acid component of the ternary complex. In this regard, a ternary complex formed between a nucleic acid polymerase, a primer / template duplex, and a labelled nucleotide during a SBS method of the disclosure will generally be unstabilised (i.e., not stabilized) to allow for the labelled nucleotide within the ternary complex to be covalently incorporated into the nascent polynucleotide strand by the polymerase. By contrast, a ternary complex formed between a nucleic acid polymerase, a primer / template duplex, and a labelled nucleotide during a SBB method of the disclosure is stabilised to prevent the labelled nucleotide within the ternary complex being incorporated into the nascent polynucleotide strand by the polymerase.Atorney Docket No.: 67649-702601

[0257] In one example, the method of sequencing a nucleic acid molecule as described herein is a method of SBS, wherein: each labelled nucleotide that binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, is subsequently incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex is further detected during incorporation of the labelled nucleotide into the nascent polynucleotide strand, and wherein the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures as the labelled nucleotides are bound to complementary nucleotides within the active site of the nucleic acid polymerase and sequentially incorporated into the nascent polynucleotide strand.

[0258] Where the method of sequencing a nucleic acid molecule is a method of SBS, each cycle comprising steps (I)-(II) may be repeated ‘n’ times, wherein ‘n’ is in the range of 2 to 1,000 or more. In this regard, the skilled person will appreciate that the number of cycles may be determined, in part, by the read length of the nucleic acid analyte. In one example, ‘n’ is less than 50. In one example, ‘n’ is in the range of 50-1000 (e.g., 50-100, or 50-150, or 50- 200, or 50-250, or 100-300, or 100-400, or 250-500, or 250-750, or 500-1000 or more). In one example, ‘n’ is greater than 1000. A skilled person will appreciate that ‘n’ may be determined and varied as needed according to the length (approximate or known length) of the nucleic acid analyte to be sequenced. In some examples, ‘n’ will be equivalent to the read length desired for the nucleic acid analyte.

[0259] In another example, the method of sequencing a nucleic acid molecule as described herein is a method of SBB, wherein: the nucleic acid analyte is primed with an oligonucleotide comprising a nucleotide at its 3’ end having a reversible blocking moiety to prevent labelled nucleotides being incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; and wherein following steps (I) and (II) in each cycle, the method further comprises the steps of:(III) performing one or more wash steps to remove labelled nucleotides;(IV) performing one or more step to remove the reversible blocker on the nucleotide at the 3’ end of the nascent polynucleotide strand, such that the nucleotide at the 3’ end ofAtorney Docket No.: 67649-702601 the nascent polynucleotide strand comprises a 3’ hydroxyl group which is capable of forming a phosphodiester bond with an adjacent nucleotide during incorporation by nucleic acid polymerase;(V) contacting the nucleic acid polymerase positioned within the area of electrical field enhancement with an unlabeled nucleotide comprising a reversible blocking moiety, wherein the unlabeled nucleotide comprises the same nucleotide species as the labelled nucleotide identified at step (II) such that the unlabeled nucleotide is incorporated by the nucleic acid polymerase into the nascent polynucleotide strand at its 3’ terminus;(VI) performing a wash step to remove unlabeled nucleotides, and(VII) repeating steps (I)-(VI) ‘n’ times to determine the order of enhanced distinct fluorescence emission signatures from labelled nucleotides as they sequentially bind to, and form ternary complexes with, complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand, wherein the sequence of the nucleic acid analyte is determined based on the order of enhanced distinct fluorescence emission signatures detected during the polymerase-based synthesis of the nascent polynucleotide strand.

[0260] Where the method of sequencing a nucleic acid molecule is a method of SBB, each cycle comprising steps (I)-(VI) may be repeated ‘n’ times, wherein ‘n’ is in the range of 2 to 100,000 or more. In this regard, the skilled person will appreciate that the number of cycles may be determined, in part, by the read length of the nucleic acid analyte. In one example, ‘n’ is less than 50. In one example, ‘n’ is in the range of 50-1000 (e.g., 50-100, or 50-150, or 50-200, or 50-250, or 100-300, or 100-400, or 250-500, or 250-750, or 500-1000). In one example, ‘n’ is in the range of 500-10000 (e.g., 500-1000, or 500-1500, or 500-2000, or 500- 2500, or 1000-3000, or 1000-4000, or 2500-5000, or 2500-7500, or 5000-10000, or 7500- 10000). In one example, ‘n’ is in the range of 5000-25000 (e.g., 5000-7500, or 7500-10000, or 10000-12500, or 12500-15000, or 15000-17500, or 17500-20000, or 20000-22500, or 22500-25000). In one example, ‘n’ is in the range of 15000-50000 (e.g., 15000-20000, or 25000-30000, or 35000-40000, or 45000-50000). In one example, ‘n’ is in the range of 50000-100000 (e.g., 50000-60000, or 60000-70000, or 70000-80000, or 80000-90000 or 90000-100000). As described herein, the skilled person will appreciate that ‘n’ may be determined and varied as needed according to the length (approximate or known length) ofAtorney Docket No.: 67649-702601 the nucleic acid analyte to be sequenced. In some examples, ‘n’ will be equivalent to the read length desired for the nucleic acid analyte.

[0261] According to certain examples of the method of sequencing a nucleic acid molecule as described herein, a single channel sequencing approach may be employed whereby a single species of labelled nucleotide is contacted with nucleic acid analyte and the polymerase at any one time, and different species of labelled nucleotide are contacted with nucleic acid analyte and the polymerase in a sequential manner. In accordance with such examples, the method of sequencing the nucleic acid molecule may be referred to as a single channel sequencing method. A method of sequencing a nucleic acid molecule using a single channel sequencing approach may be one in which: the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a single species of labelled nucleotide for a time and under conditions suitable for the labelled nucleotide to bind to a nucleotide positioned within an active site of the nucleic acid polymerase if it is complementary thereto, thereby forming a ternary complex with the complementary nucleotide and the nucleic acid polymerase; wherein:(i) the single species of labelled nucleotide is selected from the group of labelled nucleotides consisting of a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore and one or more quenchers are each linked directly or indirectly to the labelled nucleotide at (i), and are positioned relative to one another on the nucleotides such that the one or more quenchers attenuate emission intensity of the fluorophore when the fluorophore is excited,(iii) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(iv) a distinct fluorescence emission signature of the labelled nucleotide is enhanced upon binding to, and forming a ternary complex with, aAtorney Docket No.: 67649-702601 complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the presence or absence of the enhanced distinct fluorescence emission signature of the labelled nucleotide within the area of electrical field enhancement, wherein:(i) the presence of the enhanced distinct fluorescence emission signature is indicative that the labelled nucleotide has base-paired and formed a ternary complex with a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(ii) the absence of the enhanced distinct fluorescence emission signature is indicative that the labelled nucleotide has not base-paired, or not formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(III) performing one or more wash steps to remove the species of labelled nucleotide;(IV) where it is determined at (II) that the species of labelled nucleotide has not formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase, the method comprises repeating steps (I)-(III) in the presence of a different species of labelled nucleotide, wherein steps (I)-(III) are performed in repeated cycles with different species of labelled nucleotides until it is determined that one of the species of labelled nucleotides selected from the group consisting of labelled A, labelled G, labelled T, and labelled C base-pairs and forms a ternary complex with a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(V) repeating steps (I)-(IV) ‘n’ times to determine the order of enhanced distinct fluorescence emission signatures from labelled nucleotides as they sequentially bind to, and form ternary complexes with, complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand, wherein the sequence of the nucleic acid analyte is determined based on the order of enhanced distinct fluorescence emission signatures detected during the polymerase-based synthesis of the nascent polynucleotide strand.

[0262] According to another example, a method of sequencing a nucleic acid molecule using a single channel sequencing approach may be one in which:Atorney Docket No.: 67649-702601 the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a single species of labelled nucleotide in the presence of one or more quenchers for a time and under conditions suitable for the labelled nucleotide to bind to a nucleotide positioned within an active site of the nucleic acid polymerase if it is complementary thereto, thereby forming a ternary complex with the complementary nucleotide and the nucleic acid polymerase; wherein:(i) the single species of labelled nucleotide is selected from the group of labelled nucleotides consisting of a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore is linked directly or indirectly to the labelled nucleotide at (i), and wherein each fluorophore has an emission spectrum when excited which overlaps with an absorption spectrum of one or more of the quenchers,(iii) the one or more quenchers are not attached to the labelled nucleotide and are provided at a concentration that attenuates fluorescence emission intensity of the fluorophore when the fluorophore is excited;(iii) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(iv) a distinct fluorescence emission signature of the labelled nucleotide is enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the presence or absence of the enhanced distinct fluorescence emission signature of the labelled nucleotide within the area of electrical field enhancement, wherein:(i) the presence of the enhanced distinct fluorescence emission signature is indicative that the labelled nucleotide has base-paired and formed aAtorney Docket No.: 67649-702601 ternary complex with a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(ii) the absence of the enhanced distinct fluorescence emission signature is indicative that the labelled nucleotide has not base-paired, or not formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(III) performing one or more wash steps to remove the species of labelled nucleotide;(IV) where if it is determined at (II) that the species of labelled nucleotide has not formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase, the method comprises repeating steps (I)-(III) in the presence of a different species of labelled nucleotide, wherein steps (I)-(III) are performed in repeated cycles with different species of labelled nucleotides until it is determined that one of the species of labelled nucleotides selected from the group consisting of labelled A, labelled G, labelled T, and labelled C base-pairs and forms a ternary complex with a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and(V) repeating steps (I)-(IV) ‘n’ times to determine the order of enhanced distinct fluorescence emission signatures from labelled nucleotides as they sequentially bind to, and form ternary complexes with, complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand, wherein the sequence of the nucleic acid analyte is determined based on the order of enhanced distinct fluorescence emission signatures detected during the polymerase-based synthesis of the nascent polynucleotide strand.

[0263] In each of the foregoing examples describing sequential addition of single nucleotide species, each of the labelled nucleotides A, G, T and C may have a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited. Alternatively, the labelled nucleotides A, G, T and C may have the same fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited and differences between the nucleotide species are determined based on the knowledge of which nucleotide is being introduced in any one step.

[0264] In some examples, the order in which the different species of labelled nucleotides are contacted with the nucleic acid polymerase and the nucleic acid analyte is predetermined.Atorney Docket No.: 67649-702601

[0265] As described herein, a sequencing method of the disclosure may be a SBS method or a SBB method. Accordingly, the single channel sequencing method as described herein may be a SBS method or a SBB method.

[0266] In one example, the single channel sequencing method as described herein is a SBS method, wherein: each labelled nucleotide that binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, is subsequently incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex is detected during incorporation of the labelled nucleotide into the nascent polynucleotide strand; and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures as the labelled nucleotides are bound to complementary nucleotides within the active site of the nucleic acid polymerase and sequentially incorporated into the nascent polynucleotide strand.

[0267] Where the method of single channel sequencing is a method of SBS, each cycle comprising steps (I)-(IV) may be repeated ‘n’ times, wherein ‘n’ is in the range of 2 to 1,000 or more. In this regard, the skilled person will appreciate that the number of cycles may be determined, in part, by the read length of the nucleic acid analyte. In one example, ‘n’ is less than 50. In one example, ‘n’ is in the range of 50-1000 (e.g., 50-100, or 50-150, or 50-200, or 50-250, or 100-300, or 100-400, or 250-500, or 250-750, or 500-1000 or more). In one example, ‘n’ is greater than 1000. In some examples, the read length of the nucleic acid analyte is equivalent to ‘n’.

[0268] In another example, the single channel sequencing method as described herein is a SBB method, wherein: the nucleic acid analyte is primed with an oligonucleotide comprising a nucleotide at its 3’ end having a reversible blocking moiety to prevent labelled nucleotides being incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; and if it is determined at (II) that the species of labelled nucleotide has formed a ternary complex with a nucleotide within the template polynucleotide strand and the nucleic acid polymerase, the method comprises the following further steps between (III) and (IV) of any one cycle:Atorney Docket No.: 67649-702601(i) performing one or more step to remove the reversible blocker on the nucleotide at the 3’ end of the nascent polynucleotide strand, such that the nucleotide at the 3’ end of the nascent polynucleotide strand comprises a 3’ hydroxyl group which is capable of forming a phosphodiester bond with an adjacent nucleotide during incorporation by nucleic acid polymerase;(ii) contacting the nucleic acid polymerase positioned within the area of electrical field enhancement with an unlabeled nucleotide comprising a reversible blocking moiety, wherein the unlabeled nucleotide comprises the same nucleotide species as the labelled nucleotide identified at step (II) such that the unlabeled nucleotide is incorporated by the nucleic acid polymerase into the nascent polynucleotide strand at its 3’ terminus; and(iii) performing a wash step to remove all remaining unlabeled nucleotide.

[0269] Where the single channel sequencing method is a method of SBB, each cycle comprising steps (I)-(VI) may be repeated ‘n’ times, wherein ‘n’ is in the range of 2 to 100,000 or more. In this regard, the skilled person will appreciate that the number of cycles may be determined, in part, by the read length of the nucleic acid analyte. In one example, ‘n’ is less than 50. In one example, ‘n’ is in the range of 50-1000 (e.g., 50-100, or 50-150, or 50-200, or 50-250, or 100-300, or 100-400, or 250-500, or 250-750, or 500-1000). In one example, ‘n’ is in the range of 500-10000 (e.g., 500-1000, or 500-1500, or 500-2000, or 500-2500, or 1000-3000, or 1000-4000, or 2500-5000, or 2500-7500, or 5000-10000, or 7500-10000). In one example, ‘n’ is in the range of 5000-25000 (e.g., 5000-7500, or 7500-10000, or 10000- 12500, or 12500-15000, or 15000-17500, or 17500-20000, or 20000-22500, or 22500-25000). In one example, ‘n’ is in the range of 15000-50000 (e.g., 15000-20000, or 25000-30000, or 35000-40000, or 45000-50000). In one example, ‘n’ is in the range of 50000-100000 (e.g., 50000-60000, or 60000-70000, or 70000-80000, or 80000-90000 or 90000- 100000). In some examples, the read length of the nucleic acid analyte is equivalent to ‘n’.

[0270] In each of the foregoing examples describing nucleic acid sequencing methods, in order for fluorophores attached to the nucleotides to emit a fluorescence signal when colocalised with the polymerase, a source of electromagnetic energy must be provided to the sequencing reaction. Accordingly, the method further comprises providing a source of electromagnetic energy to the labelled nucleotides when co-localised with the DNA polymerase. In one example, the electromagnetic energy is provided by one or more lasers. However, other means of providing electromagnetic energy are known in the art and areAtorney Docket No.: 67649-702601 contemplated for use in the method of the disclosure. In one particular example, one or more lasers will be configured to provide electromagnetic energy (e.g., light) to the labelled nucleotides at a wavelength corresponding to the excitation wavelength of the fluorophores attached to the nucleotides.

[0271] In some examples, the method may comprise contacting the nucleic acid polymerase with the primed nucleic acid analyte in the presence of one or more synthetic labeled nucleotides, either in addition to the labelled nucleotides A, G, T and C in the mixture of labelled nucleotides, or as a substitute for one or more of labelled nucleotides A, G, T and / or C in the mixture of labelled nucleotides. In this regard, the method may further comprise detecting the enhanced distinct fluorescence emission signatures of the labelled synthetic nucleotides upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand. Accordingly, in some examples, the nucleotide of one or more labelled nucleotides in the mixture may be further selected from a synthetic nucleotide. In one example, the synthetic nucleotide is selected from 5-methylcytidine, N-methylcytidine, and N6-methyladenosine.

[0272] As described herein, the nucleic acid analyte to be sequenced is primed for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to a template polynucleotide strand. Accordingly, the method may further comprise contacting the nucleic acid analyte, with an oligonucleotide primer which is capable of hybridising specifically to a region of the template polynucleotide strand within the nucleic acid analyte to initiate synthesis by the nucleic acid polymerase.

[0273] The terms “primer” or “oligonucleotide primer” or similar as used in the context of methods of DNA and RNA sequencing shall be understood to mean an oligonucleotide which is capable of hybridising specifically to a region within a DNA or RNA molecule to initiate synthesis of a nascent DNA strand using a DNA polymerase or reverse transcriptase, as appropriate. A primer can be of any length depending on the particular technique it will be used for. For example, primers for priming synthesis of DNA in a DNA polymerase reaction (e.g., PCR) are generally between 10 and 40 nucleotides in length. One of skill could readily design a primer having a sequence of suitable complementarity to hybridise specifically to a template DNA sequence of interest using methods and software known in the art. In some examples, the oligonucleotide primer is designed to be substantially complementary to a universal adapter template sequence which has been attached to (e.g., through ligation) the 3’Atorney Docket No.: 67649-702601 end of a template DNA sequence. In other examples, the oligonucleotide primer is designed to be substantially complementary to a hairpin adapter sequences which have been attached to (e.g., through ligation) the 3’ end and 5’ end of a template DNA sequence. The use of universal primers means only one primer sequence is required to initiate DNA synthesis of any number of DNA molecules. In accordance with examples in which a library of DNA molecules or fragments there are prepared for sequencing, each of those DNA molecules or fragments thereof may have a universal adapter template sequence attached to (e.g., through ligation) the 3’ end to provide a template for a universal primer to specifically hybridise to.

[0274] Oligonucleotide primers for priming reverse transcription can be of any length depending on the particular technique they will be used for. For example, in some examples, the primers for priming synthesis of cDNA from an RNA template in a reverse transcription reaction may be between 10 and 40 nucleotides in length and have sufficient complementarity to a region within a template RNA sequence of interest such that the primer can hybridise to the template RNA sequence and initiate synthesis of a cDNA by reverse transcriptase. In other examples, a primer for priming synthesis of a cDNA from a RNA template in a reverse transcription reaction may be between 10 and 20 nucleotides in length and consist of a stretch of about 10-20 deoxythymidines that anneal to a poly(A) tail at the 3’ end of an RNA and thereby initiate synthesis of a cDNA by reverse transcriptase. The 3’ poly(A) tail may already exist in the RNA template, such as in the case of mRNA from a eukaryote. Alternatively, the 3’ poly(A) tail may be an adapter sequence which is attached to the 3’ end of the template RNA (e.g., through ligation). In other examples, the primers for priming reverse transcription may be random primers made up of a heterogeneous mixture of short oligonucleotides, typically hexamers but ranging from 6-10 nucleotides in length, with random base sequences. Due to their random binding (i.e., no template specificity), random primers can potentially anneal to any RNA species in a sample. Therefore, these primers are generally considered for reverse transcription of RNAs without poly(A) tails (e.g., rRNA, tRNA, non-coding RNAs, small RNAs, prokaryotic mRNA), degraded RNA (e.g., from FFPE tissue), and RNA with known secondary structures (e.g., viral genomes). One of skill could readily select a suitable primer type depending on the particular nucleic acid analyte and application. One of skill could also readily design a primer for use in a RNA transcription using methods and software known in the art. The term “selectively” or “specifically” as used in the context of hybridisation refers to the binding, duplexing, or hybridizing of an oligonucleotide primer to a particular (and usually predetermined) location within a template DNA or RNA sequenceAtorney Docket No.: 67649-702601(as appropriate) with a higher affinity, e.g., under more stringent conditions, than to other (non-specific) locations within a template DNA or RNA sequence, respectively. One of skill in the art will appreciate that specific hybridization between nucleotides usually relies on Watson-Crick pair bonding between complementary nucleotide sequences.

[0275] The generic term “nucleic acid polymerase” is used herein to refer to a nucleic acid synthesizing enzyme, including but not limited to, DNA polymerase, RNA polymerase, reverse transcriptase, primase and transferase. Typically, the polymerase comprises one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization may occur. The polymerase may catalyze the polymerization of nucleotides to the 3' end of a primer bound to its complementary nucleic acid strand. For example, a polymerase catalyzes the addition of a next correct nucleotide to the 3' OH group of the primer via a phosphodiester bond, thereby chemically incorporating the nucleotide to the primer. Nucleic acid polymerases may include naturally-occurring nucleic acid polymerases and any modified variations thereof, including, but not limited to, mutants, recombinants, fusions, genetic modifications, chemical modifications, synthetics, and analogs.

[0276] In accordance with examples describing methods of sequencing a nucleic acid analyte which is a DNA molecule, the nucleic acid polymerase is a DNA polymerase. As used herein, the term “DNA polymerase” refers to an enzyme that synthesizes a new strand of DNA in a 5 ' to 3 ' direction from a primer hybridized to a template strand of DNA. The DNA polymerase “reads” the template in the 3'— >5' direction, and adds individual nucleotides (bases) to the new or nascent strand in the 5 '—>3 ' direction. The polymerase requires a 3 'OH group from a primer to begin extension of a new DNA strand. Individual nucleotides (dNTPs, or dATP, dCTP, dTTP, dGTP, or A, C, T, G) are added to the extending DNA molecule iteratively as the new DNA strand is synthesized by DNA polymerase. The particular base (A, C, T, or G) depends on the sequence of the template DNA, so that the new base hybridizes to the nucleotide on the template strand through a Watson-Crick interaction. The DNA polymerase cycles between “open” and “closed” conformations. The DNA polymerase is in the open position with the primer-template DNA complex. Once an incoming nucleotide enters the active site, the polymerase cycles to the closed position.

[0277] A variety of DNA polymerases can be used, many of which are commercially- available. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. Most family A polymerases are single chain proteins that can contain multiple enzymatic functions including polymerase, 3' to 5' exonuclease activityAtorney Docket No.: 67649-702601 and 5' to 3' exonuclease activity. Family B polymerases typically have a single catalytic domain with polymerase and 3 ' to 5 ' exonuclease activity, as well as accessory factors. Family C polymerases are typically multi-subunit proteins with polymerizing and 3' to 5' exonuclease activity. In E. coh. three types of DNA polymerases have been found, DNA polymerases I, II, and III (analogous to family A, B, and C, respectively). In eukaryotic cells, three different family B polymerases, DNA polymerases a, 5, and E are implicated in nuclear replication, and a family A polymerase, polymerase y, is used for mitochondrial DNA replication. Other types of DNA polymerases include phage polymerases. Any of these polymerases, combinations of all or portions of these polymerases, as well as chimeras or hybrids between two or more of such polymerases or their equivalents can be used to form a portion or all of the DNA polymerase molecule in the method described herein.

[0278] Examples of DNA polymerases that can be used include without limitation: phi-29, Taq, T7, E. coli KI enow (from DNA pol I), E. coll DNA pol III, and Baccilus stearothermophilus (Bst) DNA pol. The DNA polymerase can also be genetically engineered, e.g., a hybrid (e.g., Phusion DNA polymerase in which a domain with strong dsDNA binding affinity is fused to a DNA polymerase to enhance processivity). Many useful DNA polymerases are commercially available (e.g., T7 DNA pol, Sequenase version 2.0™). Highly processive polymerases include phi29 and T7 DNA polymerases, and Moloney murine leukemia virus (M-MLV) reverse transcriptase. One of skill in the art will appreciate that DNA polymerases are structurally similar, and that recombinant, hybrid polymerases can be engineered using homologous domains from different polymerases.

[0279] In accordance with examples describing methods of sequencing a nucleic acid analyte which is a RNA molecule (i.e., direct sequencing of the RNA molecule), the nucleic acid polymerase may be a reverse transcriptase. As used herein, the term “reverse transcriptase” refers to any DNA polymerase that can copy and synthesize a first-strand cDNA from an RNA template. Reverse transcriptases include, but are not limited to, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from the Moloney murine leukemia virus, AMV reverse transcriptase from the avian myeloblastosis virus, and Telomerase reverse transcriptase that maintains the telomeres of eukaryotic chromosomes. Any of these reverse transcriptases, combinations of all or portions thereof, as well as chimeras or hybrids between two or more of such reverse transcriptases or their equivalentsAtorney Docket No.: 67649-702601 can be used to form a portion or all of a reverse transcriptases molecule used in a method of direct RNA sequencing as described herein.

[0280] In some examples, to avoid the potential for differences in the incorporation efficiency of fluorescently-labelled dNTPs, a nucleic acid polymerase and labels that maximize incorporation efficiency and minimize incorporation variability can be used. Several DNA polymerases can efficiently incorporate fluorescently-labelled dNTPs along DNA templates to produce very uniform fragments independent of the sequence context, and these polymerases are used in most automated Sanger / dideoxy -based methods using capillary-array DNA sequencers. A number of natural and engineered DNA polymerases are capable of incorporating fluorescently-labelled dNTPs (Reeve & Fuller Nature 376, 796-97 (1995); Rosenblum et al., Nucleic Acids Res 25, 4500-04 (1997); Ramanathan et al. Anal Biochem 337, 1-11 (2005); Aksyonov et al., Anal Biochem 348, 127-138 (2006); Tabor & Richardson, J Biol Chem 265, 8322-28 (1990); Zhu et al., Nucleic Acids Res 22, 3418-22 (1994); Zhu & Waggoner Cytometry 28, 206-211 (1997); Randolph & Waggoner Nucleic Acids Res 25, 2923-29 (1997); Mitra etal., Anal Biochem 320, 55-65 (2003); Anderson etal. Biotechniques 38, 257-264 (2005)).

[0281] As used herein, the term “labelled nucleotides” refers to any nucleotide (including naturally-occurring and non-naturally-occurring or “synthetic nucleotides”) attached directly or indirectly to a fluorophore. In accordance with examples in which the nucleotide comprises a fluorophore and quencher, the fluorophore and the quencher are both attached directly or indirectly to the nucleotide. Exemplary labelled nucleotides are described herein.

[0282] The term “nucleic acid analyte” refers to any nucleic acid molecule, the sequence of which is to be determined using the method of the disclosure. In its broadest form, a “nucleic acid” refers to a polymer having multiple nucleotide monomers. A nucleic acid analyte can be single- or double-stranded, and can be DNA (e.g., complementary DNA, genomic DNA or mitochondrial DNA), RNA, or hybrid polymers (e.g., DNA / RNA). The term “nucleic acid” does not refer to any particular length of polymer. Rather, a nucleic acid may be any length e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, greater than 10,000 bases, greater than 100,000 bases, greater than about 1,000,000 or more bases composed of nucleotides.

[0283] The nucleic acid analyte to be sequenced can be obtained from any source of interest, and can comprise DNA, mRNA, rRNA, and mimetics, analogs, and derivatives thereof. They can be isolated from cells, cell cultures, tissue samples, bodily fluids, viral samples, genomicAtorney Docket No.: 67649-702601 nucleic acid samples, cDNA preparations, environmental samples, forensic samples, or synthetic sources. Nucleic acids can be cloned, amplified, transcribed, ligated, fragmented, or otherwise manipulated according to standard methods to provide the nucleic acid to be sequenced as these manipulations do not render the nucleic acid unsuitable for subsequent sequencing as described herein. It will be understood that such nucleic acids may comprise modified, non-canonical, and / or non-natural nucleotides or nucleotide analogs, many of which are described in US12 / 945,767, which is incorporated herein by reference in its entirety for all purposes. Accordingly, the method of the disclosure may be used in a range of sequencing applications including, but not limited to, genome sequencing, transcriptome sequencing, RNA-sequencing, single-molecule sequencing, single-cell sequencing, methylation sequencing and combinations thereof.

[0284] In each of the foregoing examples, the nucleic acid analyte is sequenced by determining the order of nucleotides in a DNA molecule. In one example, the DNA is genomic DNA. In another example, the DNA is complementary DNA (cDNA) which has been derived from an RNA. In accordance with examples in which the nucleic acid analyte is cDNA, the method may comprise determining the sequence of an RNA based on the sequence for a corresponding cDNA. In other examples, the nucleic acid analyte to be sequenced is an RNA molecule, and the method comprises determining the sequence of the RNA by direct RNA sequencing e.g., using a reverse transcriptase-based approach as described in Ozsolak etal., (2009) Nature, 461 :814-818.

[0285] In each of the foregoing examples, the nucleic acid analyte may be subjected to one or more processing steps prior to being localised within the area of electrical field enhancement. These processing steps may include, but are not limited to, purification steps, reverse transcription (e.g., in the case of RNA), fragmentation to achieve desired size of template DNA, enrichment steps (e.g., for sequences or architecture of interest), depletion steps (e.g., to remove unwanted sequence), amplification steps (e.g., to increase the amount of template DNA), ligation steps (e.g., to attach adapters and other sequences of interest) and combinations thereof. The choice of processing steps and the techniques used may vary according to the application. In some examples, a combination of these processing steps may be employed to prepare one or more libraries (e.g., libraries of DNA templates) for sequencing using the method of the disclosure. The literature is replete with protocols and methods of preparing nucleic acid samples for sequencing reactions and a skilled person could readily determine the steps and methods required.Atorney Docket No.: 67649-702601

[0286] In some examples, the nucleic acid analyte is a double-stranded DNA molecule which may be denatured to produce a single stranded DNA molecule (i.e., a “target DNA sequence”, “template DNA”, or “template DNA sequence”) which can be used by the DNA polymerase enzyme to synthesize a new strand. The term “target DNA” or “template DNA” refers to a stretch of DNA which can be sequenced according to the present methods. The term encompasses both complementary strands of the target DNA molecule. One of skill will understand that the sequence of one strand of target DNA reveals the sequence of the other strand due to familiar Watson-Crick base pairing. The target DNA can be attached to one or more oligonucleotide primers or adaptors, e.g., to facilitate synthesis. However, in other examples, double-stranded DNA molecules are not denatured, but are instead retained in a double-stranded form. In some embodiments, the nucleic acid analyte is DNA. In some embodiments, the nucleic acid analyte is complementary DNA (cDNA) derived from an RNA sample.

[0287] The DNA molecules to be sequenced (including cDNA, single-stranded DNA and double-stranded DNA) may be fragmented or sheared into various sizes (0.5 kb to 10 kb) using hydrodynamic mechanical shearing with relatively narrow size distributions (Thorstenson et al., Genome Res 8, 848-855 (1998); Roe, Methods Mol Biol 255, 171-187 (2004)). Methods of fragmenting and / or shearing DNA are known in the art, including, e.g., sonication, nebulization, and enzymatic methods. Specialized devices for size-restricted DNA shearing are commercially available (e.g., Bioruptor® from Diagenode). These methods are further explicated in Sambrook (Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1989) and Ausubel (Current Protocols in Molecular Biology. New York: John Wily; 2001), which are incorporated herein by reference in their entireties for all purposes.

[0288] The nucleic acid analyte (e.g., DNA molecule) to be sequenced may be any length. For example, the nucleic acid analyte to be sequenced may be 10 or more nucleotides in length, e.g. about 20 nucleotides in length, or about 50 nucleotides in length, or about 100 nucleotides in length, or about 200 nucleotides in length, or about 300 nucleotides in length, or about 400 nucleotides in length, or about 500 nucleotides in length, or about 1000 nucleotides in length, or more (e.g., 2000, or 5000, or 10,000 or more nucleotides). In some examples, the nucleic acid analyte (e.g., DNA molecule) to be sequenced is between about 10-100000 nucleotides in length. In some examples, the nucleic acid analyte (e.g., DNA molecule) to be sequenced is between about 100-50000 nucleotides in length. In someAtorney Docket No.: 67649-702601 examples, the nucleic acid analyte (e.g., DNA molecule) to be sequenced is between about 200-20000 nucleotides in length. In some examples, the nucleic acid analyte (e.g., DNA molecule) to be sequenced is between about 400-10000 nucleotides in length. In some examples, the nucleic acid analyte (e.g., DNA molecule) to be sequenced is between about 500-5000 nucleotides in length. In some examples, the nucleic acid analyte (e.g., DNA molecule) to be sequenced is between about 500-1000 nucleotides in length. In some examples a nucleic acid analyte (e.g., DNA molecule) which is larger in size may be fragmented or sheared into various smaller sizes to facilitate sequencing. However, in other examples, a nucleic acid analyte (e.g., DNA molecule) which is larger in size may be sequenced as a single read.

[0289] The nucleic acid analytes (e.g., DNA molecules) to be sequenced may comprise one or more adapter sequences which provide a binding site for one or more oligonucleotide primers to hybridise and initiate synthesis by the nucleic acid polymerase. Accordingly, the method may comprise the further step of ligating adapters onto one or more (e.g., a library of) nucleic acid molecules (e.g., DNA molecules) to be sequenced using the method described herein. For example, the method may comprise ligating a universal adapter sequence onto the 3’ end of DNA molecules to be sequenced. In some examples, the method may comprise ligating hairpin adapters onto both ends of a double stranded DNA molecule to provide a sequencing template that comprises both the complementary and non-complementary strands of the DNA molecule in a single-stranded circular construct that can be repeatedly sequenced to provide redundant sequencing information from both strands. For single-molecule sequencing reactions, e.g., SMRT® Sequencing from Pacific Biosciences, where sequence data is generated from a single template molecule, statistical analysis of the redundant information is used to generate a consensus sequence for the target region from the single sequencing template. Further details about redundant sequencing and circular sequencing templates are provided, e.g., in US7,476,503 and US8, 153,375, both of which are incorporated herein by reference in their entireties for all purposes.

[0290] In other examples, the method may comprise ligating hairpin adapters onto only one end of a double stranded DNA molecule. Ligation of hairpin adapters onto only one end of the double stranded DNA molecule provides a sequencing template that comprises both the complementary and non-complementary strands of the DNA molecules in a single-stranded linear construct that can be sequenced to provide a sequence read from each strand. Such a template is particularly beneficial in sequencing technologies in which a single-stranded linearAtorney Docket No.: 67649-702601 template is preferred, e.g., in sequencing technologies that use a nanopore-based sensor, which have been described at length in the art and are currently being developed by several companies, including Oxford Nanopore and Genia. However, the use of template having stem-loop adapters at both ends are also contemplated for sequencing in a nanopore-based method, e.g., where the single-stranded circle produced by separating the complementary strands is used as a template for rolling-circle replication, e.g., where the nascent strand or the released phosphate groups are directed to or through the nanopore.

[0291] In each of the foregoing examples, step (II) of the method of sequencing a nucleic acid analyte comprises detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelled nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature. The enhanced distinct fluorescence emission signatures may be detected by any means known in the art for detecting fluorescence, and / or according to any of the embodiments already described herein.Area of electrical field enhancement for nucleic acid sequencing

[0292] As described herein, the nucleic acid polymerase used in the nucleic acid sequencing method is positioned within an area of electrical field enhancement. Exemplary means of producing an area of electrical field enhancement are described herein in the context of the broader method and shall be taken to apply mutatis mutandis to each and every example describing a method of sequencing a nucleic acid unless stated otherwise. For example, the area of electrical field enhancement may be provided by, for example and without limitation, surface structures (including nanostructured surfaces), confinements such as waveguides (including zero-mode waveguides) and microcavities, nanoparticles (including single nanoparticles or multiple nanoparticles, including multiple nanoparticle structures (such including nanoantennae), other metal structures, and combinations thereof.

[0293] In one example, the area of electrical field enhancement is produced by a nanophotonic confinement structure. For example, the nanophotonic confinement structure may be a zeromode waveguide (ZMW). In accordance with this example, the nucleic acid polymerase may be immobilised within the ZMW, or the nucleic acid analyte to be sequenced may be immobilised within the ZMW, to ensure that the nucleic acid polymerase is positioned within the area of electrical field enhancement during synthesis of the nascent polynucleotide strand.Atorney Docket No.: 67649-702601

[0294] In another example, the area of electrical field enhancement is produced by one or more plasmonic nanoparticles. Exemplary means of producing areas of electrical field enhancement using plasmonic nanoparticles, including the production of plasmonic hotspots using plasmonic nanoparticles positioned on plasmonic nanoantennas, are described hereinabove and shall be taken to apply mutatis mutandis to each and every example describing the method of sequencing a nucleic acid unless stated otherwise.

[0295] In certain examples, the area of electrical field enhancement is produced by a plasmonic nanoantenna as described herein. Where a plasmonic nanoantenna is employed, the nucleic acid polymerase or the nucleic acid analyte will be bound to or immobilized on the nanoscopic nucleic acid scaffold of the plasmonic nanoantenna and positioned within the region unoccupied by the nanoscopic nucleic acid scaffold. In one example, the nucleic acid polymerase is bound to or immobilized on the nanoscopic nucleic acid scaffold of the plasmonic nanoantenna to ensure that the nucleic acid polymerase is positioned within the area of electrical field enhancement during synthesis of the nascent polynucleotide strand. In one example, the nucleic acid analyte is bound to or immobilized on the nanoscopic nucleic acid scaffold (e.g., non-covalently, preferably through hydrogen bonding) to ensure that the nucleic acid polymerase is positioned within the area of electrical field enhancement during synthesis of the nascent polynucleotide strand.

[0296] For example, the plasmonic nanoantenna may comprise:(i) two plasmonic nanoparticles;(ii) a nanoscopic nucleic acid scaffold; and(iii) a nucleic acid polymerase; wherein:(a) the two plasmonic nanoparticles are bound to the nanoscopic nucleic acid scaffold and positioned relative to one another such that a plasmonic hotspot exists between the plasmonic nanoparticles,(b) the plasmonic hotspot comprises a region unoccupied by the nanoscopic nucleic acid scaffold,(c) the nucleic acid polymerase is bound to the nanoscopic nucleic acid scaffold and is positioned within the region (of the plasmonic hotspot) unoccupied by the nanoscopic nucleic acid scaffold, and optionally, a nucleic acid analyte bound to the nanoscopic nucleic acid scaffold.Atorney Docket No.: 67649-702601

[0297] The plasmonic nanoparticles may be formed of any material which possesses plasmonic resonance properties. Suitable plasmonic nanoparticles are described hereinabove and include, but are not limited to, metallic nanoparticles, metal alloy nanoparticles, polymeric nanoparticles and derivatives and composites thereof. In one particular example, each of the plasmonic nanoparticles are independently selected from metallic nanoparticles.|0298] As previously described herein, the plasmonic nanoparticles may be formed in any suitable or desired shape and size. For example, the plasmonic nanoparticles may be selected independently from nanospheres, nanorods, nanoprisms, nanocubes, nanoshells, nanotubes, or nanostars. The plasmonic nanoparticles may be the same shape or they may be heterogeneous. In one example, the plasmonic nanoparticles are nanospheres.

[0299] In addition to shape, a skilled person will appreciate that the size of the nanoparticles in the plasmonic nanoantenna may vary. In some examples, the plasmonic nanoparticles may have the same, or substantially the same, largest diameter. For example, each of the plasmonic nanoparticles may have a largest diameter in the range of about 5 nm to about 500 nm (e.g., in the range of about 10 nm to about 250 nm, or in the range of about 20 nm to about 200 nm, or in the range of about 50 nm to about 150 nm, or in the range of about 80 nm to about 120 nm). In some examples, each of the plasmonic nanoparticles has a largest diameter of about 80 nm. In some examples, each of the plasmonic nanoparticles has a largest diameter of about 100 nm. In some examples, each of the plasmonic nanoparticles has a largest diameter of about 120 nm.

[0300] Whereas in other examples the largest diameter may differ between plasmonic nanoparticles on the same plasmonic nanoantenna. In accordance with an example in which the largest diameter differs between plasmonic nanoparticles on the same plasmonic nanoantenna, the plasmonic nanoparticles may have an average largest diameter in the range of about 5 nm to about 500 nm. For example, the plasmonic nanoparticles on the plasmonic nanoantenna may have an average largest diameter in the range of about 10 nm to about 250 nm (e.g., . in the range of about 20nm to about 200nm, or about 30 nm to about 200 nm, or about 40 nm to about 200 nm, or about 50 nm to about 200 nm, or about 60 nm to about 200 nm, or about 70 nm to about 200 nm, or about 80 nm to about 200 nm, or about 90 nm to about 200 nm, or about 100 nm to about 200 nm, or about 50 nm to about 150 nm, or about 75 nm to about 150 nm, or about 100 nm to about 120 nm, or about 100 nm to about 150 nm) In one example, the average diameter of the nanoparticles is about 100 nm. In one particular example, the plasmonic nanoparticles are gold or gold-coated nanoparticles (e.g.,Atorney Docket No.: 67649-702601 nanospheres) having an average diameter of about 100 nm. In another example, the plasmonic nanoparticles are silver or silver-coated nanoparticles (e.g., nanospheres) having an average diameter of about 100 nm.

[0301] As described herein, the plasmonic nanoantenna of the disclosure comprises two plasmonic nanoparticles, each attached to the nanoscopic nucleic acid scaffold and positioned relative to one another such that a plasmonic hotspot exists between the plasmonic nanoparticles. However, in some examples, the plasmonic nanoantenna may comprise more than two plasmonic nanoparticles (e.g., 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 or more nanoparticles), each attached to the nanoscopic nucleic acid scaffold and positioned relative to one another such that a plasmonic hotspot exists between the plasmonic nanoparticles collectively. In accordance with examples in which the plasmonic nanoantenna may comprises more than two plasmonic nanoparticles, two or more of the plasmonic nanoparticles may be aggregated.

[0302] The plasmonic nanoparticles are attached to the nanoscopic nucleic acid scaffold and positioned relative to one another such that a plasmonic hotspot comprising a region unoccupied by the nanoscopic nucleic acid scaffold exists between the plasmonic nanoparticles. As used herein, the phrase “region unoccupied by nanoscopic nucleic acid scaffold”, or similar, shall be understood to refer to the region or space within the plasmonic hotspot which is accessible to the nucleic acid polymerase e.g., when bound to the nanoscopic nucleic acid scaffold. This unoccupied region may also be referred to as “cleared space” within the plasmonic hotspot or a “cleared hotspot”. Preferably, the “region unoccupied by nanoscopic nucleic acid scaffold” is sufficiently large that, when a nucleic acid polymerase is positioned therein, the enzymatic activity of the polymerase is retained. Said another way, the enzymatic activity of the polymerase is not lost due to steric effects (e.g., blockade) of the nanoscopic nucleic acid scaffold. Methods of measuring and / or determining the dimensions and / or volume of a plasmonic hotspot between at least two plasmonic particles and cleared space which is accessible to analytes are known in the art, for example, as described in Close etal., (2022) Advanced Materials Interfaces, 9(24), with specific reference to Supplementary Figure 12 (S12). For example, in the case of a plasmonic nanoantenna described herein where the cleared space takes the form of a cube with the dimensions L=W=H=30 nm, the cleared volume of the hotspot may be calculated as 2.7e4 nmA3 = 2.7e-20 L = ~27 zL, wherein 1 nmA3 is equal to le-24 L and 1 zL is equal to le-21 L. In the case of a plasmonic nanoantenna described herein where the cleared space takes the form of a sphere with a radius (r) of 15 nm,Atorney Docket No.: 67649-702601 the cleared volume may be calculated as 4 / 37irA3 = ~14zL, wherein 1 nmA3 is equal to le-24 L and 1 zL is equal to le-21 L. The skilled person would also appreciate that the dimension(s) and / or volume of the region unoccupied by the nanoscopic nucleic acid scaffold can be determined based on the volume of the nanoscopic nucleic acid scaffold that overlaps with the volume and / or dimensions of the plasmonic hotspot. A skilled person may also take the following factored into account when designing a plasmonic nanoparticle having a hotspot of sufficient size and intensity, including the dimensions and design of the nanoscopic nucleic acid scaffold, wavelength of incident light polarisation of incident light, propagation of incident light, material of nanoparticles, shape of nanoparticles, volume of nanoparticles, interparticle distance, number of nanoparticles, relative position of nanoparticles, dielectric constant of surrounding medium and / or wavelength(s) of emission detection

[0303] In some examples, the region within the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold is defined by a three-dimensional space having a length (L) between the two plasmonic nanoparticles, a height (H) at a midpoint (L / 2) that is perpendicular to (L), and a width (W) at L / 2 that is perpendicular to L and H, wherein at L / 2, W is selected from about 20 nm to about 100 nm (e.g., selected from about 30 nm to about 100 nm, or about 40 nm to about 100 nm or about 50 nm to about 100 nm, or about 20 nm to about 50 nm or about 30 nm to about 50 nm) and H is selected from about 10 nm to about 100 nm (e.g., selected from about 30 nm to about 100 nm, or about 40 nm to about 100 nm or about 50 nm to about 100 nm, or about 20 nm to about 50 nm or about 30 nm to about 50 nm), and wherein L is measured at the shortest distance between the two plasmonic nanoparticles. The skilled person will appreciate that the shape of the nanoparticle (examples of which are described herein), and the geometry of the nucleic acid scaffold, can impact the height and width of the plasmonic hotspot, as well as the height (H) and width (W) of the region within the plasmonic hotspot which is unoccupied. The skilled person will appreciate that the interparticle distance of the plasmonic nanoparticles when attached to the plasmonic nanoantenna may be varied (or “tuned”) to alter the size and magnitude of the plasmonic hotspot, as well as the region unoccupied by the nanoscopic nucleic acid scaffold. As used herein, the term “interparticle distance” refers to the distance between any two plasmonic nanoparticles on the nanoscopic DNA scaffold of the plasmonic nanoantenna In some examples, the interparticle distance between the plasmonic nanoparticles L is about lOnm to about lOOnm (e.g., selected from about 10 nm to about 80 nm, or about 20 nm to about 80 nm, or about 30 nm to about 80 nm, or about 40 nm to about 80 nm, or about 50 nm to aboutAtorney Docket No.: 67649-70260180 nm). In another example, the interparticle distance between the plasmonic nanoparticles L is about 20 nm to about 100 nm (e.g., selected from about 20 nm to about 50 nm, or about 30 nm to about 50 nm, or about 20 nm to about 40 nm, or about 30 nm to about 40 nm). In one example, the plasmonic nanoparticles have an interparticle distance L of about 20nm to about 50nm. In one particular example, the plasmonic nanoparticles have an interparticle distance L of about 30 nm. In certain examples, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold has a volume of at least about 1 zL, such as from about IzL to about 10 zL, or from about 5zL to about 10 zL. In certain examples, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold has a volume which is greater than or equal to about 10 zL. In some examples, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold has a volume of at least about lOzL (e.g., at least about 15zL, or at least about 20zL, or at least about 25zL, or at least about 30zL, or at least about 35zL, or at least about 40zL, or at least about 50zL). For example, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold may have a volume of about lOzL to about 50zL (e.g., selected from about lOzL to about 50zL, or from about 20zL to about 50zL, or from about 30zL to about 50zL, or from about 40zL to about 50zL, or from about lOzL to about 40zL, or from about 20zL to about 40zL, or from about 30zL to about 40zL, or from about lOzL to about 30zL, or from about 20zL to about 30zL, or from about 40zL to about 50zL). In one example, the region of the plasmonic hotspot which is unoccupied by the nanoscopic nucleic acid scaffold as a volume of about 20zL to about 40zL.

[0304] Alternatively, or in addition, the region of the plasmonic hotspot unoccupied by the nanoscopic nucleic acid scaffold may be expressed as a relative percentage of the total volume of the plasmonic hotspot. In some examples, the region unoccupied by the nanoscopic nucleic acid scaffold is 70% or more (e.g., 75%, or 80%, or 85% or 90% or 95% or more) of the plasmonic hotspot.

[0305] The skilled person will appreciate that the plasmonic hotspot, as well as the region of the plasmonic hotspot unoccupied by the nanoscopic nucleic acid scaffold, may take different shapes or form e.g., depending on the choice of nanoparticles and geometry of the nucleic acid scaffold. In one example, the region unoccupied by the nanoscopic nucleic acid scaffold may be amorphous. Alternatively, the region unoccupied by the nanoscopic nucleic acid scaffold may have a substantially defined shape. For example, the region unoccupied by the nanoscopic nucleic acid scaffold may be spherical, elliptical, cubic or cuboid.Atorney Docket No.: 67649-702601

[0306] The plasmonic nanoparticles may be immobilised at predetermined positions on the surface of the nanoscopic nucleic acid scaffold via hybridisation between (i) DNA (i.e., oligonucleotides) coating the plasmonic nanoparticles, and (ii) DNA (i.e., polynucleotides) located at predetermined positions and extending from the surface of the nanoscopic nucleic acid scaffold which are complementary or substantially complementary to the DNA coating the plasmonic nanoparticles i.e., the nanoparticles may be immobilised covalently via Watson-crick base-paring). In this way, the interparticle distance of the plasmonic nanoparticles on the surface of the nanoscopic nucleic acid can be controlled by appropriate design and positioning of the polynucleotides extending from the surface of nanoscopic nucleic acid scaffold.

[0307] In some examples, the plasmonic nanoparticles may be coated with, or conjugated / linked to, a plurality of oligonucleotides capable of hybridising to polynucleotides extending from the surface of the nanoscopic nucleic acid scaffold. For example, each plasmonic nanoparticle may be coated with a plurality of DNA oligonucleotides selected from 2 to about 100 oligonucleotides (e.g., from 2 to about 75 oligonucleotides, or from 2 to about 50 oligonucleotides, or from 2 to about 25 oligonucleotides, or from 2 to about 10 oligonucleotides). In other examples, each plasmonic nanoparticle may be coated with 5 or more DNA oligonucleotides (e.g., 6, or 7, or 8, or 9 or 10 or 11, or 12, or 13, or 14, or 15 or more DNA oligonucleotides. Preferably, the DNA oligonucleotide coating substantially covers the surface of each of the plasmonic nanoparticles. For example, the DNA oligonucleotide coating may cover about 75% or more (e.g., selected from about 80%, or about 85% or about 90% or about 95% or more) of the surface of each of the plasmonic nanoparticles.

[0308] In addition to facilitating immobilization of the coated plasmonic nanoparticles to the surface of the nanoscopic nucleic acid scaffold, the DNA oligonucleotides coated on the plasmonic nanoparticles reduces aggregation of the plasmonic nanoparticles relative to plasmonic nanoparticles without the coating during preparation of the plasmonic nanoantenna. Exemplary methods of measuring aggregation of nanoparticles are known in the art, including but not limited to, UV-visible spectroscopy, differential centrifugal sedimentation, particle counters and / or a dynamic light-scattering, particle tracking analysis and single-particle inductively coupled plasma mass spectrometry. These exemplary methods are described in Minelli et al., (2019) Langmuir, 35(14):4927-4935. In one example, the coating of DNA oligonucleotides reduces aggregation of the plasmonic nanoparticles by 35% or more (e.g.,Atorney Docket No.: 67649-70260140%, or 45%, or more) relative to plasmonic nanoparticles without the coating. In one example, the coating of DNA oligonucleotides reduce aggregation of the plasmonic nanoparticles by 50% or more (e.g., 55%, or 60%, or more) relative to plasmonic nanoparticles without the coating. In one example, the coating of DNA oligonucleotides reduce aggregation of the plasmonic nanoparticles by 65% or more (e.g., 70%, or 75%, or more) relative to plasmonic nanoparticles without the coating.

[0309] The coating or conjugation of the one or more DNA molecules (e.g., oligonucleotides) to the plasmonic nanoparticles may be performed by any method known in the art. However, in one example, the one or more oligonucleotides is bound to the plasmonic nanoparticles by a linker. In one particular example, the oligonucleotides may be thiolated in accordance with the Examples disclosed herein. However, it will be appreciated that other functional anchor groups, such as -COOH, -NH2, and -OH, may be used to covalently attach DNA oligonucleotides to the surface of nanoparticles.

[0310] The DNA oligonucleotides coating the plasmonic nanoparticles are designed such that their nucleotide sequences are complementary or substantially complementary to sequences of the cognate DNA polynucleotides extending from the surface of the nanoscopic nucleic acid scaffold and enable hybridisation therebetween. In certain examples, more than 50% of the nucleotides (e.g., more than 50%, or more than 60%, or more than 70%, or more than 80% of the nucleotides) in the oligonucleotide or plurality of oligonucleotides coating the plasmonic nanoparticles is selected from pyrimidine nucleotides (i.e., thymine and cytosine). In some examples, each nucleotide in the oligonucleotide or plurality of oligonucleotides coating the plasmonic nanoparticles is selected from pyrimidine nucleotides (i.e., thymine and cytosine).

[0311] The DNA oligonucleotide attached to the plasmonic nanoparticles, and the complementary or substantially complementary DNA polynucleotide within, or extending from, the nanoscopic nucleic acid scaffold, may be modified or unmodified. Suitable modifications are known in the art. They include for example Azo-modifications known in the art which can be used to realize photoresponsive and reversible binding and unbinding of the respective DNA molecules. Further modifications include modifications that modulate the strength of the interaction between the respective DNA molecules, such as backbone modifications that alter the surface charge (e.g. peptide nucleic acids (PNA)) or structural flexibility to induce hybridization efficiency (e.g. locked nucleic acids (LNA)). Even further modifications include the use of photocleavable structures known in the art.Atorney Docket No.: 67649-702601

[0312] As described herein, the plasmonic nanoantenna comprises a nanoscopic nucleic acid scaffold onto which (i) the plasmonic nanoparticles and (ii) the nucleic acid polymerase molecule or nucleic acid analyte, are immobilised or bound. The nanoscopic nucleic acid scaffold may be a nanoscopic DNA scaffold. The nanoscopic DNA scaffold may comprise or be formed of one or more DNA origami structures. The term “DNA origami structures” and similar shall be understood to refer to scaffolds or structures formed by DNA molecules which are self-assembling and produced by the so called DNA-origami technique. Scaffolded DNA origami is a technique which was introduced in 2006 by Paul Rothemund, building on the earlier approach of structural DNA nanotechnology, and provided access to finite DNA nanoobjects. In this technique, single-stranded circular DNA molecules of typically 7.25 kb size are folded by the aid of staple strands into an array of helices through an arrangement of periodic crossovers. Extension of this concept to assemble 3D DNA structures has led to the establishment of a robust and reliable method to fabricate DNA nanostructures (or “nanoscopic DNA scaffolds”) with dimensions in the 20 to 100 nm range. However, larger structures are possible. DNA origami structures can be a bundle of tubes or pipes, e.g. 3-, 6- , or 12-helix bundles, or can have a substantially flat, rectangular shape. In this way, the DNA origami structures of varying size and dimensions can be combined (e.g., stacked or layered) to construct a nanoscopic DNA scaffold of any particular shape. Origami structures can be used as "molecular pegboards" with an addressable surface area of a few thousand nm2for arranging arbitrary objects-of-interest (OOI) with a "single pixel" resolution of about 6 nanometers. Furthermore, the shape, dimensions and contour of the surface of origami structures may be tailored such that the nanoscopic DNA scaffold has a surface which is geometrically suitable for attaching molecules of interest (e.g., the plasmonic nanoparticles and polymerase as described herein. Methods for generating DNA origami structures, including those having DNA sequence strands protruding from the structure, are known in the art. In one example, nanoscopic DNA scaffold comprises one or more 3D DNA origami structures. In certain example examples, at least a portion of the DNA origami structures are assembled to form a U-shaped or curved structure.|0313] In accordance with an example in which the nanoscopic DNA scaffold is assembled in a U-shape, the nanoscopic DNA scaffold will comprise a first face to which one of two plasmonic nanoparticles is anchored via a nucleic acid linker and a second face to which a second of two plasmonic nanoparticles is anchored via a nucleic acid linker. The first face ofAtorney Docket No.: 67649-702601 the scaffold and the second face of the scaffold may be on the same or adjacent sides, or on opposite sides, of the nanoscopic DNA scaffold.

[0314] In one example, the nanoscopic DNA scaffold will comprise (i) one or more DNA polynucleotides extending from the first face of the scaffold, each comprising a sequence which is complementary to a sequence of one or more DNA oligonucleotides coating one of the two plasmonic nanoparticles, and (ii) one or more DNA polynucleotides extending from the second face of the scaffold, each comprising a sequence which is complementary to a sequence of one or more DNA oligonucleotides coating one of the two plasmonic nanoparticles. The DNA polynucleotides forming part of, or extending from, the nanoscopic DNA scaffold, may each be or comprise a single-stranded DNA. The DNA polynucleotides extending from the first and second faces of the scaffold will each comprise a region of singlestranded DNA which is of sufficient length and complementarity to the DNA oligonucleotides coating the plasmonic nanoparticles to permit hybridisation. Upon hybridisation of a cognate DNA polynucleotide and DNA oligonucleotides to form a nucleic acid linker, a plasmonic nanoparticle comprising to DNA oligonucleotide is anchored to the surface of the nanoscopic DNA scaffold. Each nucleic acid linker anchoring a plasmonic nanoparticle to the nanoscopic DNA scaffold will be formed by one of the DNA oligonucleotides coated on the surface of the plasmonic nanoparticles hybridised to a DNA polynucleotide forming part of, or extending from, the nanoscopic DNA scaffold. In one example, the DNA polynucleotides extending from the first and second faces of the scaffold comprise a M13mpl8-derived scaffold sequence and the DNA oligonucleotides coating the plasmonic nanoparticles comprises a staple sequence which is complementary thereto.

[0315] The length of the DNA polynucleotides extending from the first and second faces of the scaffold may be varied as desired. However, in certain examples, the DNA polynucleotides extending from the first and second faces of the scaffold each comprise about 20 nucleotides to about 50 nucleotides (e.g., from about 20 nucleotides to about 40 nucleotides, or about 20 nucleotides to about 30 nucleotides, or about 30 nucleotides to about 50 nucleotides, or about 30 nucleotides to about 50 nucleotides). In one example, the DNA polynucleotides extending from the first and second faces of the scaffold comprise about 20 nucleotides to about 30 nucleotides.

[0316] In certain examples, each plasmonic nanoparticles is anchored to the nanoscopic DNA scaffold via multiple nucleic acid linkers (e.g., via 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 or more nucleic acid linkers). It will be appreciated that the number of nucleic acid linkersAtorney Docket No.: 67649-702601 connecting each plasmonic nanoparticle positively correlates with the binding strength of the plasmonic nanoparticle to the nanoscopic DNA scaffold. Accordingly, the number of DNA polynucleotides extending from the nanoscopic DNA scaffold, and which hybridise to DNA oligonucleotides coated on the plasmonic nanoparticles, can be chosen freely to accomplish a desired binding strength and / or a desired hybridization efficiency depending from the particular application of interest. Furthermore, the length of the region of Watson-Crick pairing in the nucleic acid linker (z.e., between the DNA polynucleotide(s) extending from the nanoscopic DNA scaffold and the DNA oligonucleotide(s) attached to the plasmonic nanoparticles) can be varied to accomplish a desired binding specificity and / or binding strength and / or a desired hybridization efficiency, depending from the particular application of interest. In certain examples, the region of Watson-Crick pairing is from about 8-30 nucleotides in length, or between about 10-25 nucleotides in length, or about 12-20 nucleotides in length. The terms “hybridise”, “hybridising” or similar as used herein refers to a process whereby two complementary or substantially complementary nucleic acid sequences anneal to each in accordance with Watson-Crick base pairing rules.

[0317] The skilled person will appreciate that the DNA oligonucleotides attached to the plasmonic nanoparticles, and the complementary or substantially complementary DNA polynucleotide within, or extending from, the nanoscopic DNA scaffold (which together hybridise to form the nucleic acid linkers that anchor the plasmonic nanoparticles to the nanoscopic DNA scaffold), may be of any length. For example, the respective DNA molecules may have lengths independently selected from between about 20 to about 50 nucleotides in length or between about 20 to about 40 nucleotides in length, or between about 20 to about 30 nucleotides in length. Preferably, the region of Watson-Crick pairing between the DNA polynucleotide sequence(s) extending from the nanoscopic nucleic acid scaffold (e.g., DNA origami structures) and the DNA oligonucleotide sequences attached to the plasmonic nanoparticles is from about 8-30 nucleotides in length, or between about 10-25 nucleotides in length, or about 12-20 nucleotides in length, e.g.

[0318] The terms “hybridise”, “hybridising” or similar as used herein refers to a process whereby two complementary or substantially complementary nucleic acid sequences anneal to each in accordance with Watson-Crick base pairing rules.

[0319] As used herein, the term "substantially complementary" refers to two sequences that are fully complementary or that are partially complementary to one another, such that they will hybridize under appropriately stringent hybridization conditions. Said another way, theAtorney Docket No.: 67649-702601 term "substantially complementary” is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between two nucleic acid sequences e.g., between the DNA sequences attached to the plasmonic nanoparticles, and the complementary or substantially complementary DNA sequences within the nanoscopic DNA scaffold. It is understood that the sequence of a nucleic acid need not be 100% complementary to that of its target or complement (although this may be one preferred example). For example, the sequence of a nucleic acid may be at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% complementary to the sequence of its target or complement.

[0320] In some embodiments, the nucleic acid polymerase is immobilised within the unoccupied area (or ‘cleared space’) of the plasmonic hotspot formed by the plasmonic nanoantenna described herein, whilst at the same time retaining the biological activity of the nucleic acid polymerase, e.g., immobilisation of the nucleic acid polymerase to the surface of the nanoscopic nucleic acid scaffold is achieved by a nucleic acid linker. In one example, the nucleic acid linker which tethers the nucleic acid polymerase to the surface of the nanoscopic nucleic acid scaffold (e.g., the nanoscopic DNA scaffold described herein) is a doublestranded DNA linker comprising (i) a DNA polynucleotide which is covalently or non- covalently bound to the nucleic acid polymerase via an amino acid within the nucleic acid polymerase, and (ii) a DNA polynucleotide forming part of, or extending from, the surface of the nanoscopic nucleic acid scaffold, wherein the polynucleotides at (i) and (ii) each comprise single-stranded DNA sequences which are complementary or substantially complementary to one another and are capable of hybridizing to one another. The polynucleotide bound to the nucleic acid polymerase may comprise a peptide tag, which is in turn bound to nucleic acid polymerase via an isopeptide bond formed between an amino acid within the nucleic acid polymerase and an amino acid within the peptide tag. In the present case, the inventors employed a SpyCatcher-SpyTag bioconjugation system as described in Hatlem et al (2019) hit. J. Mol. Set, 20(9) :2129 to attach the DNA polynucleotide of the double-stranded DNA linker to the nucleic acid polymerase. In accordance with an example in which a SpyCatcher- SpyTag system is used, the polymerase is modified to express a SpyCatcher protein on its surface. The cognate SpyTag is then conjugated to the polynucleotide by any means known in the art. Methods of producing oligonucleotide-peptide conjugates are known in the art, such as described in Klabenkova et al (2021) Molecules, 26(17):5420, and include (but are not limited to) conjugation through thioether or disulfide bond, native ligation, oxime linkage,Atorney Docket No.: 67649-702601 thiazolidine linkage or hydrazone linkage, amine bond formation, click chemistry, Diels- Alder reaction, and thiol-maleimide conjugation. However, in one particular example, the DNA polynucleotide is modified to comprise a maleimide and the SpyTag is conjugated to the DNA polynucleotide through thiol-maleimide conjugation (e.g., as described in the Examples herein). Once the DNA polynucleotide comprising the SpyTag is produced, it can be contacted with the polymerase modified to express a SpyCatcher protein. Upon recognition, the SpyCatcher and SpyTag form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag, such that the polymerase and the DNA polynucleotide of the double-stranded DNA linker are bound. Although the SpyCatcher-SpyTag system was used herein, the skilled person would appreciate that alternative enzyme-mediate conjugation systems (including variant Catcher-Tag systems) may be employed and may rely on alternative amino acid combinations known for forming isopeptide bonds to covalently bind the polymerase and the DNA polynucleotide of the double-stranded DNA linker. The skilled person would also be aware of alternative chemistries for protein ligation and bioconjugation which may be employed and these are contemplated herein.

[0321] In some examples, the amino acid within the polymerase to which the polynucleotide linker is bound is located with the N-terminal domain of the polymerase.

[0322] According to other examples, the nucleic acid analyte to be sequenced is bound to, or immobilized on, the surface of the nanoscopic nucleic acid scaffold within the unoccupied area (or ‘c...

Claims

Atorney Docket No.: 67649-702601CLAIMS:

1. A method of detecting binding of a labelled species to an analyte of interest, comprising:(I) contacting one or more labelled species, each comprising a fluorophore, with the analyte of interest in the presence of one or more quenchers of the fluorophore(s) and within an area of electrical field enhancement, wherein:(i) the one or more labelled species comprises one or more quenchers, and the fluorophore and one or more quenchers are positioned relative to one another on the labelled species such that the one or more quenchers attenuate fluorescence emission intensity of the fluorophore(s) when the fluorophore is excited; and / or(ii) the one or more quenchers are provided separately and at a concentration that attenuates fluorescence emission intensity of the fluorophore(s) when the fluorophore is excited, wherein the labelled species has a distinct fluorescence emission signature which is enhanced when the fluorophore is excited within the area of electrical field enhancement; and(II) detecting the enhanced distinct fluorescence emission signature of the labelled species within the area of electrical field enhancement, following excitation of the fluorophore with light at a wavelength corresponding to an excitation wavelength of the fluorophore, wherein detection of the enhanced distinct fluorescence emission signature indicates that the labelled species is bound to the analyte of interest.

2. The method of claim 1, wherein the distinct fluorescence emission signatures of the one or more labelled species are enhanced within the area of electrical field enhancement relative to the distinct fluorescence emission signatures of corresponding labelled species outside of the area of electrical field enhancement at equivalent fluorophore concentrations.Atorney Docket No.: 67649-7026013. The method of claim 1 or claim 2, wherein the signal to noise ratio (SNR) of the one or more labelled species is increased within the area of electrical field enhancement compared to performance of the method using the corresponding labelled species without the one or more quenchers at equivalent fluorophore concentrations.

4. The method of any one of claims 1 to 3, wherein background noise of the one or more labelled species outside the area of electrical field enhancement is reduced compared to the background noise of the one or more labelled species outside the area of electrical field enhancement when the method is performed using the corresponding labelled species without the one or more quencher at equivalent fluorophore concentrations.

5. The method of any one of claims 1 to 4, wherein at least one or each fluorophore attached to the one or more labelled species is present at a concentration of at least about lOnM, optionally between about lOnM and about lOOnM.

6. The method of any one of claims 1 to 5, wherein at least one or each fluorophore attached to the one or more labelled species has a fluorescence lifetime of at least about 0.1 ns, optionally between about 0.1 ns and about 500 ns.

7. The method of any one of claims 1 to 6, wherein at least one or each fluorophore attached to the one or more labelled species has a peak emission wavelength between about 350 nM and about 850 nM, optionally between about 350 nM and about 650 nM.

8. The method of any one of claims 1 to 7, wherein at least one or each fluorophore attached to the one or more labelled species has a Stokes shift of greater than or equal to about 5 nm, optionally between about 5nm to about 30nm.

9. The method of any one of claims 1 to 8, wherein at least one or each fluorophore attached to the one or more labelled species has a quantum yield greater than about 50%.Atorney Docket No.: 67649-70260110. The method of any one of claims 1 to 9, wherein at least one or each fluorophore attached to the one or more labelled species is an organic fluorophore.

11. The method of any one of claims 1 to 10, wherein at least one or each fluorophore attached to the one or more labelled species is independently selected from the group consisting of: fluorescein, rhodamine, cyanine, dipyrromethene, naphthalene, xanthene, anthracene, squaraine and triangulenium.

12. The method of any one of claims 1 to 11, wherein at least one or each fluorophore attached to the one or more labelled species is independently selected from the group consisting of: ATTO 565, ATTO 655, Acridine Orange, Acridine Yellow, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, BODIPY 500 / 510, BODIPY 530 / 550, BODIPY FL, BODIPY TR-X, Cascade Blue, Coumarin 6, CY2, CY3B, CY3, CY3.5, CY5, CY5.5, Dansyl, DAPI, DPH, Erythrosin, Ethidium Bromide, Fluorescein, FITC, FURA-2, GFP, Hoechst 33258, Hoechst 33342, HPTS, Indocyanine Green, KU530, KU560, Laurdan, Lucifer Yellow, Nile Red, Oregon Green, Prodan, Pyrene, Rhodamine 101, Rhodamine 110, Rhodamine 123, Rhodamine 6G, Rhodamine B, Ru(bpy)3[PFe]2, Ru(bpy)2(dcpby)[PFe]2, SeTau dyes such as SeTau-380, SeTau-404, SeTau-405, SeTau-647, SeTau-425, SITS, SNART, Stilbene SITS, SIT A, Texas Red, TOTO-1, YOYO-1, and YOYO-3.

13. The method of any one of claims 1 to 12, wherein the one or more quenchers are independently selected from the group consisting of: BHQ-0, BHQ-1, BHQ-2, BHQ- 3, BHQ-10, QSY7, QSY9, QSY21, QSY35, Dabeyl, Dabsyl, Cy5Q, Cy7Q, DYQ-660, DYQ-661, 540Q, 580Q, and 612Q.

14. The method of any one of claims 1 to 13, wherein the area of electrical field enhancement is produced by a plasmonic nanostructure.Atorney Docket No.: 67649-70260115. The method of claim 14, wherein the plasmonic nanostructure comprises one or more plasmonic nanoparticles, a nanostructured surface, or a nanophotonic confinement structure (optionally a zero-mode waveguide or microcavity).

16. The method of any one of claims 1 to 14, wherein the area of electrical field enhancement is produced by a plasmonic hotspot defined between two or more plasmonic nanoparticles.

17. The method of claim 16, wherein the plasmonic hotspot is defined between the two or more plasmonic nanoparticles located on a plasmonic nanoantenna.

18. The method of claim 17, wherein the plasmonic nanoantenna comprises two plasmonic nanoparticles and a nanoscopic nucleic acid scaffold, wherein:(a) the two plasmonic nanoparticles are bound to the nanoscopic nucleic acid scaffold and positioned relative to one another such that a plasmonic hotspot exists between the two plasmonic nanoparticles; and(b) the plasmonic hotspot comprises a region unoccupied by the nanoscopic nucleic acid scaffold in which binding of the one or more labelled species to the analyte of interest occurs.

19. The method of any one of claims 1 to 18, wherein the distinct fluorescence emission signatures of the labelled species are distinguishable based on differences in peak emission wavelength, differences in fluorescence emission intensity, differences in fluorescence emission lifetime, differences in duration between successive fluorescence emissions, or any combinations thereof.

20. The method of claim 19, wherein two or more labelled species comprise different fluorophores, and the enhanced distinct fluorescence emission signatures of the respective labelled species are distinguished based on distinct peak emission wavelengths of the different fluorophores.

21. The method of claim 20, wherein the distinct peak emission wavelengths are separated by 10 nm or more relative to each other.Atorney Docket No.: 67649-70260122. The method of any one of claims 1 to 21, wherein two or more labelled species comprise the same fluorophore, and the enhanced distinct fluorescence emission signatures of the respective labelled species are distinguished based on differences in fluorescence emission intensity and / or fluorescence emission lifetime.

23. The method of any one of claims 1 to 22, wherein detecting the enhanced distinct fluorescence emission signature in step (II) is performed using fluorescence microscopy or a fluorometer.

24. The method of any one of claims 1 to 23, wherein detecting the enhanced distinct fluorescence emission signatures in step (II) is performed using total internal reflection fluorescence (TIRF) microscopy.

25. The method of any one of claims 1 to 24, which is a method of sequencing a nucleic acid molecule, wherein: the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a mixture of labelled nucleotides for a time and under conditions such that one of the labelled nucleotides binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, wherein the complementary nucleotide is positioned within an active site of the nucleic acid polymerase; wherein:(i) the mixture of labelled nucleotides comprises a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore and one or more quenchers are each linked directly or indirectly to the respective nucleotides, and are positioned relative to one another on the nucleotides such that the one or more quenchersAtorney Docket No.: 67649-702601 attenuate emission intensity of the fluorophore when the fluorophore is excited,(iii) each of the labelled nucleotides A, G, T and C has a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited,(iv) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(v) the distinct fluorescence emission signatures of the labelled nucleotides are enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelled nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature; wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the labelled nucleotides sequentially bind to, and form ternary complexes with, the complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand.

26. The method of any one of claims 1 to 24, which is a method of sequencing a nucleic acid molecule, wherein: the or each labelled species is a labelled nucleotide; the or each labelled species is a labelled nucleotide; the analyte of interest is a nucleic acid analyte which is primed and provides a template polynucleotide strand for polymerase-based synthesis of a nascent polynucleotide strand which is complementary to template polynucleotide strand of the nucleic acid analyte; step (I) comprises contacting a nucleic acid polymerase with the nucleic acid analyte and a mixture of labelled nucleotides in the presence of one or more quenchersAtorney Docket No.: 67649-702601 for a time and under conditions such that one of the labelled nucleotides binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, wherein the complementary nucleotide is positioned within an active site of the nucleic acid polymerase; wherein:(i) the mixture of labelled nucleotides comprises a labelled adenine nucleotide (A), a labelled guanosine nucleotide (G), a labelled thymine nucleotide (T), and a labelled cytosine nucleotide (C),(ii) a fluorophore is linked directly or indirectly to each of the nucleotides, wherein each fluorophore has an emission spectrum when excited which overlaps with an absorption spectrum of one or more of the quenchers,(iii) the one or more quenchers are not attached to the labelled nucleotides and are provided at a concentration that attenuates fluorescence emission intensity of the fluorophores when the fluorophores are excited,(iv) each of the labelled nucleotides A, G, T and C has a distinct fluorescence emission signature when the fluorophore to which the respective nucleotide is linked is excited,(iv) the nucleic acid polymerase is positioned within the area of electrical field enhancement, and(v) the distinct fluorescence emission signatures of the labelled nucleotides are enhanced upon binding to, and forming a ternary complex with, a complementary nucleotide within the template polynucleotide strand and the nucleic acid polymerase; and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex, and determining the identity of the labelled nucleotide within the ternary complex based on its enhanced distinct fluorescence emission signature;Atorney Docket No.: 67649-702601 wherein steps (I) and (II) are repeated ‘n’ times and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the labelled nucleotides sequentially bind to, and form ternary complexes with, complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand.

27. The method of claim 25 or 26, wherein the method of sequencing the nucleic acid analyte is a method of sequencing by synthesis.

28. The method of claim 27, wherein: each labelled nucleotide that binds to a complementary nucleotide within the template polynucleotide strand and forms a ternary complex with the complementary nucleotide and the nucleic acid polymerase, is subsequently incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; the enhanced distinct fluorescence emission signature of the labelled nucleotide within the ternary complex is detected during incorporation of the labelled nucleotide into the nascent polynucleotide strand; and the sequence of the nucleic acid analyte is determined by determining the order of enhanced distinct fluorescence emission signatures as the labelled nucleotides are bound to complementary nucleotides within the active site of the nucleic acid polymerase and sequentially incorporated into the nascent polynucleotide strand.

29. The method of claim 25 or 26, wherein the method of sequencing the nucleic acid analyte is a method of sequencing by binding.

30. The method of claim 29, wherein: the nucleic acid analyte is primed with an oligonucleotide comprising a nucleotide at its 3’ end having a reversible blocking moiety to prevent labelled nucleotides being incorporated into the nascent polynucleotide strand by the nucleic acid polymerase; and wherein following step (II), the method further comprises the steps of:(III) performing one or more wash steps to remove labelled nucleotides;Atorney Docket No.: 67649-702601(IV) performing one or more step to remove the reversible blocker on the nucleotide at the 3’ end of the nascent polynucleotide strand, such that the nucleotide at the 3’ end of the nascent polynucleotide strand comprises a 3’ hydroxyl group which is capable of forming a phosphodiester bond with an adjacent nucleotide during incorporation by nucleic acid polymerase;(V) contacting the nucleic acid polymerase positioned within the area of electrical field enhancement with an unlabeled nucleotide comprising a reversible blocking moiety, wherein the unlabeled nucleotide comprises the same nucleotide species as the labelled nucleotide identified at step (II) such that the unlabeled nucleotide is incorporated by the nucleic acid polymerase into the nascent polynucleotide strand at its 3’ terminus;(VI) performing a wash step to remove all remaining unlabeled nucleotide, and(VII) repeating steps (I)-(VI) ‘n’ times to determine the order of enhanced distinct fluorescence emission signatures from labelled nucleotides as they sequentially bind to, and form ternary complexes with, complementary nucleotides within the template polynucleotide strand and the nucleic acid polymerase, during polymerase-based synthesis of the nascent polynucleotide strand, wherein the sequence of the nucleic acid analyte is determined based on the order of enhanced distinct fluorescence emission signatures detected during the polymerase-based synthesis of the nascent polynucleotide strand.

31. The method of any one of claims 25 to 30, wherein the nucleotide of each labelled nucleotide is further independently selected from a synthetic nucleotide, optionally independently selected from 5-methylcytidine, N-methylcytidine, or N6- methyladenosine.

32. The method of any one of claims 25 to 31, wherein the nucleic acid analyte is a DNA molecule and / or the nucleic acid polymerase is a DNA polymerase.

33. The method of claim 32, wherein the DNA molecule is a complementary DNA (cDNA) molecule derived from an RNA, optionally wherein the method comprisesAtorney Docket No.: 67649-702601 determining the sequence of the RNA based on the sequence for the corresponding cDNA.

34. The method of any one of claims 25 to 31, wherein the nucleic acid analyte is a RNA molecule and the nucleic acid polymerase is a RNA transcriptase.

35. The method of any one of claims 25 to 34, wherein the fluorophore of the labelled nucleotide is directly or indirectly linked to the nucleotide via a 5’ phosphate or 5’ polyphosphate of the nucleotide.

36. The method of claim 25 or any one of claims 27 to 35 when appended to claim 25, wherein the fluorophore and the quencher of the fluorophore are both indirectly linked to the nucleotide the via a 5’ phosphate or 5’ polyphosphate of the nucleotide.

37. The method of claim 35 or 36, wherein the polyphosphate is a tri-, tetra-, penta- or hexa-phosphate.

38. The method of any one of claims 35 to 37, wherein the fluorophore is bound to a terminal phosphate of the polyphosphate.

39. The method of claim 25 or any one of claims 27 to 38 when appended to claim 25, wherein the labelled nucleotide is a compound of formula (I):A-!_R„„A2™R2„„.A3(Formula I) wherein:R1is or comprises the fluorophore;R2is or comprises the quencher;A1and A3are each independently absent or is;A2is absentAtorney Docket No.: 67649-702601L1to L3are each independently absent or a divalent linking moiety which is uninterrupted or interrupted, and optionally substituted;L4is absent or is a terminal moiety which is uninterrupted or interrupted, and optionally substituted;L5is absent or is a trivalent linking moiety which is uninterrupted or interrupted, and optionally substituted; each X is independently absent or is a nucleotide, on the proviso that at least one X is present; wherein R1and R2are optionally interchangeable.

40. The method of claim 39, wherein the labelled nucleotide is a compound of any one of formulae (II) to (IV):Formula (IV), wherein:X is a nucleotide.

41. The method of claim 39 or claim 40, wherein the labelled nucleotide is a compound of any one of formula (Ila)-(IIf):Atorney Docket No.: 67649-702601Formula (Ila) Formula (lib)Formula (lie) Formula (Ilf) wherein:B1is a nucleobase; n is 1 to 5;R3is H, Me, an alcohol protecting group, or a 3’ blocker; and R9is H, OH, or OMe.

42. The method of claim 39 or claim 40, wherein the labelled nucleotide is a compound of any one of formula (Illa)-(IIId):Formula (Illa) Formula (Illb)Atorney Docket No.: 67649-702601Formula (IIIc) Formula (Hid) wherein:B1is a nucleobase; n is 1 to 5;R3is H, Me, an alcohol protecting group, or a 3’ blocker; and R9is H, OH, or OMe.

43. The method of claim 39 or claim 40, wherein the labelled nucleotide is a compound of any one of formula (IVa)-(IVd):Formula (IVc) Formula (IVd) wherein:B1is a nucleobase;Atorney Docket No.: 67649-702601 n is 1 to 5;R3is H, Me, an alcohol protecting group, or a 3’ blocker; andR9is H, OH, or OMe.

44. The method of any one of claims 39 to 43, wherein L1to L5are each independently absent or an aliphatic group which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, -C(=O)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, - S(=O)2-, -N(R4)C(=S)N(R4)-, and -N(R4)C(=O)N(R4)-, and is optionally substituted with one or more R5, wherein: each R4is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, alkylcarbocyclyl, and alkylheterocyclyl, each of which is optionally substituted with one or more R5; each R5is independently selected from the group consisting of H, halogen, Ci- walkyl, OCi-ioalkyl, Ci-iohaloalkyl, OCi-iohaloalkyl, C2-ioalkenyl, C2-ioalkynyl, OC2- walkenyl, OC2-ioalkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, Ci-ioalkyl-3-10-membered-carbocyclyl, Ci-ioalkyl-3-10-membered-heterocyclyl, - NO2, -CN, -SCN, -N3, =0, -N(R5)2, -C(=O)N(R5)2, -S(=O)N(R5)2, -S(=O)2N(R5)2, - OR5, -SR5, OC(=O)R5, -C(=O)R5, -C(=O)OR5-S(=O)R5, -S(=O)2R5, -S(=O)OR5, - S(=O)2OR5, -S(=O)(OR5)2, -OS(=O)R5, -OS(=O)2R5, -OS(=O)OR9, -OS(=O)2OR5, - OS(=O)(OR5)2, -N(R5)C(=O)R5, -N(R5)S(=O)R5, N(R5)C(=O)N(R5)2, -N(R5)S(=O)2R5, -P(=O)(OR5)2, -P(=O)OR5(R5), -P(=O)(R5)2, -OP(=O)(OR5)2, - OP(=O)OR5(R5) and -OP(=O)(R5)2, wherein each Ci-ioalkyl, Ci-iohaloalkyl, C2- walkenyl, C2-ioalkynyl, 3-10 membered-carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R6; each R6is independently selected from the group consisting of H, Ci-ealkyl, 3- 10 membered carbocyclyl, 3-10-membered heterocyclyl, Ci-6alkyl-3-10-membered- carbocyclyl, and Ci-6alkyl-3-10-membered-heterocyclyl; wherein each Ci-ealkyl, 3-10- membered-carbocyclyl, and 3-10-membered heterocyclyl is optionally substituted with one or more R7;Atorney Docket No.: 67649-702601 each R7is independently selected from the group consisting of H, halogen, -NO2, -N(R8)2, -CN, -SCN, -N3, =0, -C(=0)R8, -C(=0)0R8, -C(=O)N(R8)2, -N(R8)C(=O)R8, -OR8, -P(=O)(OR8)2, -P(=O)OR8(R8), -P(=O)(R8)2, Ci-6alkyl, and -OCi-6alkyl; and each R8is independently selected from the group consisting of H, Ci-ioalkyl, Ci- walkyl, C2-ioalkenyl, C2-ioalkynyl, 3-10 membered carbocyclyl, 3-10 membered heterocyclyl, Ci-ioalkyl-3-lO-membered carbocyclyl, Ci-ioalkyl-3-lO-membered heterocyclyl.

45. The method of any one of claims 39 to 44, wherein L1to L5are each independently absent or is Ci-3oalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, -C(=0)NR4-, -NR4-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -N(R4)C(=S)N(R4)-, and -N(R4)C(=O)N(R4)-, and is optionally substituted with one or more R5.

46. The method of any one of claims 39 to 45, wherein L1to L5are each independently absent or is Ci-2oalkyl which is uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, -C(=0)NH-, -C(=O)O-.

47. The method of any one of claims 1 to 24, which is a method of sequencing a polypeptide, wherein: the analyte of interest is a polypeptide analyte which is positioned within the area of electrical field enhancement, each labelled species is a labelled amino acid recognition molecule which is capable of selectively associating with at least one type of terminal amino acid of the polypeptide, and the labelled amino acid recognition molecule comprises the fluorophore and the one or more quenchers, step (I) comprises contacting the polypeptide analyte with the one or more labelled amino acid recognition molecules and a cleaving reagent for a time and under conditions such that at least one labelled amino acid recognition molecule selectively associates with a terminal amino acid of the polypeptide in the presence of the cleavage reagent,Atorney Docket No.: 67649-702601 wherein selective association of the one or more labelled amino acid recognition molecules with the terminal amino acid and subsequent cleavage of the terminal amino acid from the polypeptide analyte, results in a distinct fluorescence emission signature for each type of terminal amino acid when the fluorophore to which the respective amino acid recognition molecule is linked is excited, wherein the distinct fluorescence emission signature for each type of terminal amino acid is enhanced within the area of electrical field enhancement; and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the terminal amino acid when selectively associated with its cognate amino acid recognition molecule and subsequently cleaved from the polypeptide terminus, and determining the identity of the terminal amino acid based on its enhanced distinct fluorescence emission signature, wherein steps (I)-(II) are repeated ‘n’ times and the sequence of the polypeptide analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the amino acid types exposed at the polypeptide terminus are sequentially cleaved from the polypeptide by the cleavage reagent, thereby providing the sequence of the amino acid types in the polypeptide analyte.

48. The method of any one of claims 1 to 24, which is a method of sequencing a polypeptide, wherein: the analyte of interest is a polypeptide analyte which is positioned within the area of electrical field enhancement, each labelled species is a labelled amino acid recognition molecule which is capable of selectively associating with at least one type of terminal amino acid of the polypeptide, and wherein the labelled amino acid recognition molecule comprises the fluorophore, step (I) comprises contacting the polypeptide analyte with one or more of the labelled amino acid recognition molecule(s), a cleaving reagent and one or more quenchers for a time and under conditions such that at least one labelled amino acidAtorney Docket No.: 67649-702601 recognition molecule selectively associates with a terminal amino acid of the polypeptide in the presence of the cleavage reagent and the one or more quenchers, wherein the one or more quenchers are not attached to the amino acid recognition molecule(s) and are provided at a concentration that attenuates fluorescence emission intensity of the fluorophore(s) when the or each fluorophore is excited, wherein selective association of the one or more labelled amino acid recognition molecules with the terminal amino acid and subsequent cleavage of the terminal amino acid from the polypeptide analyte, results in a distinct fluorescence emission signature for each type of terminal amino acid when the fluorophore to which the respective amino acid recognition molecule is linked is excited, wherein the distinct fluorescence emission signature for each type of terminal amino acid is enhanced within the area of electrical field enhancement; and step (II) comprises detecting the enhanced distinct fluorescence emission signature of the terminal amino acid when selectively associated with its cognate amino acid recognition molecule and subsequently cleaved from the polypeptide terminus, and determining the identity of the terminal amino acid based on its enhanced distinct fluorescence emission signature, wherein steps (I)-(II) are repeated ‘n’ times and the sequence of the polypeptide analyte is determined by determining the order of enhanced distinct fluorescence emission signatures detected as the amino acid types exposed at the polypeptide terminus are sequentially cleaved from the polypeptide by the cleavage reagent, thereby providing the sequence of the amino acid types in the polypeptide analyte.

49. The method of claim 47 or claim 48, wherein the labelled amino acid recognition molecule and the cleavage reagent are the same molecule.

50. The method of claim 47 or claim 48, wherein the labelled amino acid recognition molecule and the cleavage reagent are different molecules.Atorney Docket No.: 67649-70260151. The method of any one of claims to 47 to 50, wherein one or more labelled amino acid recognition molecules is capable of reversibly associating with the terminal amino acid of the polypeptide.

52. The method of any one of claims 47 to 51, wherein one or more or each of the labelled amino acid recognition molecules is capable of binding selectively and with affinity (KD) to one type of amino acid, optionally wherein the binding affinity of the or each labelled amino acid recognition molecule for its cognate type of amino acid is at least about 50 nM or higher.

53. The method of any one of claims 47 to 52, wherein each labelled amino acid recognition molecule selectively binds to one type of amino acid, wherein: the one type of amino acid is an amino-terminal amino acid; the one type of amino acid is a carboxy -terminal amino acid; the one type of amino acid is one of the twenty naturally occurring amino acids or a subset of types thereof; the one type of amino acid is a modified variant of one of the twenty naturally occurring amino acids or a subset of unmodified and / or modified variants thereof; or the one type of amino acid is selected from amino acids with charged side chains (e.g, positively and / or negatively charged side chains), amino acids with polar side chains (e.g., polar uncharged side chains), amino acids with nonpolar side chains (e.g, nonpolar aliphatic and / or aromatic side chains), and amino acids with hydrophobic side chains.

54. The method of any one of claims 47 to 53, wherein the polypeptide analyte is contacted with a plurality of labelled amino acid recognition molecules, each selectively associating with a different type of amino acid and each having a distinct fluorescence emission signature.Atorney Docket No.: 67649-70260155. The method of any one of claims 47 to 54, wherein each labelled amino acid recognition molecule which selectively associates with a distinct type of amino acid is linked to a fluorophore having a distinct fluorescence emission signature which can be distinguished from fluorescence emission signatures of other labelled amino acid recognition molecules, optionally wherein each labelled amino acid recognition molecule which selectively associates with a distinct type of amino acid is linked to a fluorophore having a distinct peak emission wavelength and / or a distinct fluorescence emission intensity and / or a distinct fluorescence emission lifetime.

56. The method of any one of claims 1 to 24, which is a method of detecting the presence of an analyte of interest in a sample, wherein: step (I) comprises contacting a capture reagent positioned within the area of electrical field enhancement with the sample and the labelled species, wherein the capture reagent and the labelled species are each independently capable of binding to the analyte of interest, the fluorophore and quencher are positioned relative to one another on the labelled species such that the quencher attenuates the fluorophore’ s emission intensity when the fluorophore is excited irrespective of structural conformation of the labelled species, and detecting the enhanced distinct fluorescence emission signature of the labelled species at step (II) indicates that the labelled species is bound to the analyte of interest, indicating the presence of the analyte of interest in the sample.

57. The method of claim 56, wherein the labelled species is a compound of formula (I): 1™R1~A2™R2„A3(Formula I) wherein:R1is or comprises the fluorophore;R2is or comprises the quencher;Atorney Docket No.: 67649-702601A1and A3are each independently absent oris;A2is absentL1to L3are each independently absent or a divalent linking moiety which is uninterrupted or interrupted, and optionally substituted;L4is a terminal moiety which is uninterrupted or interrupted, and optionally substituted;L5is a trivalent linking moiety which is uninterrupted or interrupted, and optionally substituted; each X is independently absent or comprises an analyte binding moiety, on the proviso that at least one X is present; wherein R1and R2are optionally interchangeable.

58. The method of claim 57, wherein the labelled species is a compound of any one of formulae (II) to (IV):R ' L1- X- L2-R2Formula (II)Formula (III)L3....Rr.....L5.....R2......L3XFormula (IV), wherein X is present and comprises the analyte binding moiety.

59. The method of claim 57, wherein:(i) the labelled species is a compound of formulae (II):Atorney Docket No.: 67649-702601R - - L1X I2-R2Formula (II) wherein X is present and comprises the analyte binding moiety, and one or both of L1and L2are optionally absent; or(ii) the labelled species is a compound of any one of formulae (III):Formula (III). wherein:X is present and comprises the analyte binding moiety;L3is absent;R1is a labelled nucleotide comprising a nucleotide covalently linked to the quencher, wherein the nucleotide is selected from the group consisting of A, G, T or C; andL1is a polynucleotide sequence comprising and / or consisting of between about 1 to 30 contiguous nucleotides (optionally between about 1 to 20 nucleotides).

60. The method of any one of claims 57 to 59, wherein the analyte binding moiety is selected from the group consisting of: small molecule, peptide, polypeptide, protein and nucleic acid.

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