Plasma nano antenna and application thereof in single molecule sequencing

Abstract

The present disclosure generally relates to methods of sequencing nucleic acid molecules, such as DNA, more particularly single DNA molecules, using polymerases located within electric field enhanced regions. The present disclosure also relates to plasma nanoantennas useful in nucleic acid sequencing, methods of making the nanoantennas, arrays comprising the nanoantennas, and methods of using the nanoantennas and arrays in single molecule sequencing techniques.

Description

Relevant application data

[0001] This application claims priority to Australian Provisional Patent Application No. 2023901389, filed on 8 May 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to methods for sequencing nucleic acid molecules, plasma nanoantennas, methods for preparing said nanoantennas, arrays containing said nanoantennas, and methods for using said nanoantennas and arrays in single-molecule sequencing technologies. Background Technology

[0003] DNA sequencing reads the sequence of four nucleotide bases—adenine (A), thymine (T), cytosine (C), and guanine (G)—arranged into long-chain biopolymers that encode the genes of all living organisms. The ability to sequence DNA has revolutionized bioscience and is having a rapid and profound impact on multiple industries, including medicine, agriculture, and biotechnology. The DNA sequencing market is projected to reach $60 billion by 2030. However, the current wave of industry-leading high-throughput sequencing methods still faces many significant challenges, including the need for large amounts of DNA, limitations on the maximum length of DNA that can be read sequentially, and the computational resources required to construct sequences from raw data. Therefore, a prominent goal is to read the sequence of individual DNA molecules accurately and in real time. Summary of the Invention

[0004] In recent years, single-molecule sequencing technology has made significant progress, with two emerging technologies—Oxford Nanopore's "current-blocking" sequencing and PacBio's "zero-mode waveguide" sequencing—currently leading the market. Both companies are valued at approximately $2 billion each. However, the methods used in both technologies are fundamentally limited by our ability to precisely manipulate individual DNA molecules and restrict the observation volume to a single nucleotide. These limitations lead to a high error rate, thus requiring multiple repeated sequencing reactions to achieve statistically accurate sequencing. Therefore, there is a need for true single-molecule DNA sequencing.

[0005] Metal nanoparticles and plasmas can serve as auxiliary antennas to enhance molecular fluorescence, for example, in the field of optical sensors. By coupling electromagnetically radiating molecules with the plasma of nanoparticles, the strength and dynamics of the interaction between molecules and light can be influenced, thereby increasing the emission rate of fluorescent molecules. Therefore, nanoparticle-based plasmon resonance sensing is a label-free method that has been developed for detecting the presence of analytes in samples and / or for real-time studies of biomolecular interactions.

[0006] This document provides plasmon resonance-based sensing methods and compositions for improving DNA molecule sequencing. The plasmon resonance-based sensing methods and compositions provided herein also include solutions to challenges previously unconsidered or unknown in the development of plasmon resonance-based sensing methods for sequencing. While previous attempts have been made to apply plasmon resonance-based sensing to the single-molecule level of complex biomolecules such as proteins (e.g., US20130252825A1), plasmon resonance-based sensing methods and compositions for efficient sequencing reactions have not been experimentally validated, and / or remain challenging, particularly due to the difficulty in immobilizing large biomolecules (such as enzymes) within hotspots formed by nanoparticles, thereby preserving the function of these biomolecules (e.g., polymerases). Therefore, there is a need for new methods and tools for analyzing single molecules using nanoparticle-based plasmon resonance methods. This disclosure is partly based on the inventors' recognition that, although plasmon nanoantennas have been developed and proven for a variety of applications, previous attempts to apply plasmon nanoantennas to biological applications have been limited by the types of molecules that can be tethered to a DNA scaffold within a “plasmon hotspot” that defines the region of electric field enhancement. The limitations of existing plasma nanoantenna technology stem in part from the fact that the plasma hotspot size required for effective signal enhancement is relatively small, which limits the size of molecules that can bind within the hotspot and the size of analytes that can enter the hotspot during use. While recent efforts have produced plasma nanoantennas with so-called “cleared space” within the plasma hotspot, there has been no evidence to date that enzymes bind within the plasma hotspot of a plasma nanoantenna, let alone that enzymes bound within the hotspot can maintain their activity despite the steric hindrance caused by the limited space within the DNA origami structure. However, the inventors have now developed a plasma nanoantenna comprising a nanoscale DNA scaffold with two plasma nanoparticles anchored to it to form a plasma hotspot therebetween, and have demonstrated for the first time that an active protein (i.e., DNA polymerase) can be anchored within the unoccupied region (or “cleared space”) within the plasma hotspot. The inventors have also experimentally demonstrated that when DNA polymerase is anchored within the plasma hotspot designed in their nanoantenna, it retains its biological function; and that the plasma hotspot, while containing DNA polymerase, maintains a sufficient electric field strength to enhance the fluorescence signal. In this regard, the inventors have demonstrated that the light intensity emitted by fluorophores connected to or co-located with protein molecules within the hot spot can be enhanced compared to the light intensity emitted by corresponding fluorophores outside the hot spot.However, the most important finding of the inventors is the following experimental discovery: when DNA polymerase is tethered within the plasma hotspot of its nanoantenna in the presence of fluorescently labeled deoxynucleoside triphosphates (dNTPs) and a DNA template, the sequence in which the DNA polymerase incorporates specific fluorescently labeled dNTPs into the elongating DNA strand during DNA synthesis can be detected by measuring the enhanced and distinctive fluorescence signals emitted from the corresponding dNTPs upon incorporation into the plasma hotspot. Therefore, the inventors have demonstrated that the plasma nanoantenna of this disclosure can be used for single-molecule DNA sequencing applications.

[0007] Therefore, in one instance, this disclosure provides a method for sequencing nucleic acid analytes, the method comprising: (I) Under certain time and conditions, a nucleic acid polymerase is contacted with a nucleic acid analyte and a labeled nucleotide, such that the labeled nucleotide is sequentially incorporated by the polymerase into a polynucleotide having a sequence complementary to the polynucleotide sequence of the nucleic acid analyte, wherein the polymerase is located within an electric field-enhanced region; and each labeled nucleotide comprises: (i) Adenine nucleotide (A), guanine nucleotide (G), thymine nucleotide (T) or cytosine nucleotide (C). (ii) Fluorescein, and (iii) Polyphosphate linkers that bind nucleotides to fluorophores; In this group, A, G, T, and C are each independently linked to the fluorophore via polyphosphate. When the fluorophore linked to the corresponding nucleotide is excited, each of the labeled nucleotides A, G, T, and C exhibits a unique fluorescence emission characteristic, and The unique fluorescence emission characteristics of the labeled nucleotides are enhanced when incorporated by nucleic acid polymerase into a sequence complementary to the polynucleotide sequence of the nucleic acid analyte; and (II) The sequence of the nucleic acid analyte is determined by detecting the sequence of enhanced unique fluorescence emission features when the labeled nucleotides are sequentially incorporated by a nucleic acid polymerase into a polynucleotide having a sequence complementary to the polynucleotide sequence of the nucleic acid analyte.

[0008] In one instance, the fluorescence emission characteristics of the labeled nucleotides were not enhanced outside the region where the electric field was enhanced.

[0009] In one example, the method includes contacting a nucleic acid polymerase with a nucleic acid analyte in the presence of each of the labeled nucleotides A, G, T, and C.

[0010] In some instances, the nucleotide of the labeled nucleotide may be further selected from synthetic nucleotides. In one instance, the synthetic nucleotide is selected from 5-methylcytidine, N-methylcytidine, and N6-methyladenosine.

[0011] The synthetic nucleotide is preferably linked to a fluorophore via a polyphosphate group. According to this method, an example of a synthetic nucleotide linked to a fluorophore via a polyphosphate group is used, where, when the fluorophore linked to the corresponding nucleotide is excited, each of the nucleotides A, G, T, and C, as well as each of the synthetic nucleotides, exhibits a unique fluorescence emission characteristic.

[0012] In one instance, the unique enhanced fluorescence emission feature of the labeled nucleotide is increased by two times or more (e.g., about three times or more, or about four times or more, or about five times or more, or about six times or more, or about seven times or more, or about eight times or more, or about nine times or more) compared to the unique fluorescence emission feature of the corresponding labeled nucleotide outside the field-enhanced region. In some instances, the enhanced unique fluorescence emission feature of the labeled nucleotide is increased by an order of magnitude or more (e.g., about 10 times or more, or about 20 times or more, or about 30 times or more, or about 40 times or more, or about 50 times or more, or about 60 times or more, or about 70 times or more, or about 80 times or more, or about 90 times or more, or about 100 times or more) compared to the unique fluorescence emission feature of the corresponding labeled nucleotide outside the field-enhanced region. In other instances, the enhanced unique fluorescence emission feature of the labeled nucleotide is increased by at least about 100 times or more (e.g., at least about 150 times, or at least about 200 times, or at least about 250 times, or at least about 300 times, or at least about 350 times, or at least about 400 times, or at least about 450 times, or at least about 500 times, or at least about 550 times, or at least about 600 times, or at least about 650 times, or at least about 700 times, or at least about 750 times, or at least about 800 times, or at least about 850 times, or at least about 900 times, or at least about 950 times, or at least about 1000 times) compared to the unique fluorescence emission feature of the corresponding labeled nucleotide outside the electric field enhancement region.

[0013] The unique fluorescence emission characteristics described herein can be distinguished from each other based on differences in peak emission wavelength, fluorescence emission intensity, fluorescence emission duration, fluorescence lifetime, duration between consecutive fluorescence emissions, or any combination thereof.

[0014] In one instance, the peak emission wavelength of each fluorophore is independently selected from emission wavelengths in the visible spectrum, ultraviolet (UV) spectrum, infrared (IR) spectrum, and near-infrared spectrum. In another instance, the peak emission wavelength of each fluorophore is independently selected from emission wavelengths between approximately 350 nm and 850 nm. In some instances, one or more fluorophores or each fluorophore has a low quantum efficiency (or low quantum yield).

[0015] In one instance, the unique fluorescence emission characteristics of two or more types of labeled nucleotides can be distinguished based on differences in peak emission wavelengths. In one instance, each different type of nucleotide is associated with a fluorophore having a unique peak emission wavelength. In any instance where the labeled nucleotides can be distinguished based on differences in peak emission wavelengths, the peak emission wavelengths of the labeled nucleotides are 10 nm or more apart from each other. For example, the peak emission wavelengths may be about 25 nm or more apart from each other. For example, the peak emission wavelengths may be about 50 nm or more apart from each other. For example, the peak emission wavelengths may be about 75 nm or more apart from each other. For example, the peak emission wavelengths may be about 100 nm or more apart from each other.

[0016] In one instance, the unique fluorescence emission characteristics of two or more types of labeled nucleotides can be distinguished based on differences in fluorescence lifetime.

[0017] Alternatively or additionally, two or more types of nucleotides are attached to the same fluorophore, and the different types of nucleotides are distinguished based on differences in fluorescence emission intensity and / or fluorescence emission duration. In one example, the different types of nucleotides are distinguished based on differences in fluorescence emission intensity. In another example, the different types of nucleotides are distinguished based on differences in fluorescence emission duration.

[0018] As described herein, each labeled nucleotide used in the methods of this disclosure comprises a nucleotide linked to a fluorophore via a polyphosphate. In one instance, the polyphosphate is a triphosphate, tetraphosphate, pentaphosphate, or hexaphosphate. For example, the polyphosphate is a triphosphate. For example, the polyphosphate is a tetraphosphate. For example, the polyphosphate is a pentaphosphate. For example, the polyphosphate is a hexaphosphate.

[0019] In some instances, the fluorophore binds to the phosphate group furthest from the nucleotide.

[0020] In some instances, the method involves contacting a nucleic acid polymerase with a nucleic acid analyte and labeled nucleotides in the presence of one or more quenchers.

[0021] In one example, the method includes providing an electromagnetic wave source to an electric field-enhancing region, wherein the wavelength of the electromagnetic wave is sufficient to excite a fluorophore linked to a labeled nucleotide.

[0022] In each of the above examples, the steps for sequentially detecting the enhanced unique fluorescence emission feature at (II) using a fluorescence microscope or fluorometer are as follows: For example, using a total internal reflection fluorescence (TIRF) microscope to sequentially detect the enhanced unique fluorescence emission feature at (II). For example, using a confocal microscope to sequentially detect the enhanced unique fluorescence emission feature at (II). For example, using an epifluorescence microscope to sequentially detect the enhanced unique fluorescence emission feature at (II).

[0023] In each of the above examples, the nucleic acid analyte can be DNA. In one example, the DNA is genomic DNA. In another example, the DNA is complementary DNA (cDNA) derived from RNA. Depending on the example where the nucleic acid analyte is cDNA, the method may include determining the RNA sequence based on the sequence of the corresponding cDNA.

[0024] In some instances, the method further includes contacting the DNA with an oligonucleotide primer that is capable of hybridizing specifically with a region of the DNA, thereby initiating synthesis by a nucleic acid polymerase (e.g., DNA polymerase). In some instances, the region of the DNA hybridized by the oligonucleotide primer is an oligonucleotide adaptor, and the oligonucleotide primer is substantially complementary to the oligonucleotide adaptor. In some instances, the DNA to be sequenced contains an oligonucleotide adaptor at its 3' end. For example, DNA containing an oligonucleotide adaptor at its 3' end can be single-stranded DNA. For example, DNA containing an oligonucleotide adaptor at its 3' end can be double-stranded DNA. In other instances, the DNA to be sequenced is a double-stranded DNA flanked by two hairpin loops, wherein the hairpin loops are formed of single-stranded DNA, and at least one of the hairpin loops contains an oligonucleotide adaptor. The method may also include ligating the oligonucleotide adaptor to the DNA to be sequenced prior to the contact step in (I).

[0025] In other instances, the DNA to be sequenced is single-stranded DNA, and its 3' end contains a hairpin oligonucleotide adaptor, wherein the hairpin oligonucleotide adaptor contains internally complementary sequences capable of forming a hairpin structure, the hairpin structure containing a double-stranded region for initiating synthesis by a nucleic acid polymerase (e.g., DNA polymerase). The method may also include ligating the hairpin oligonucleotide adaptor to the DNA to be sequenced prior to the contact step in (I).

[0026] In each of the above examples, the nucleic acid analyte can be a single molecule. Therefore, the method can include contacting a nucleic acid polymerase with a single nucleic acid molecule (e.g., a single DNA molecule).

[0027] In each of the above examples, the electric field enhancement region can be generated by a plasma hotspot, which is generated by a plasma nanoantenna. The plasma nanoantenna may comprise at least two plasma nanoparticles.

[0028] In a specific example, the plasmonic nanoantenna used to generate the electric field enhancement region in this method comprises: Two plasma nanoparticles; Nanoscale nucleic acid scaffolds; and Nucleic acid polymerase; in: (a) Two plasma nanoparticles are attached to a nanoscale nucleic acid scaffold and positioned relative to each other, resulting in a plasma hotspot between the two plasma nanoparticles; (b) Plasma hotspots include regions not occupied by nanoscale nucleic acid scaffolds, and (c) The nucleic acid polymerase binds to the nanoscale nucleic acid scaffold and is located in a region not occupied by the nanoscale nucleic acid scaffold, and (d) Optionally, nucleic acid analytes are bound to nanoscale nucleic acid scaffolds.

[0029] This disclosure also provides a plasma nanoantenna comprising: Two plasma nanoparticles; Nanoscale nucleic acid scaffolds; and Nucleic acid polymerase; in: (a) Two plasma nanoparticles bind to a nanoscale nucleic acid scaffold and are positioned relative to each other, resulting in plasma hotspots between the two plasma nanoparticles. (b) Plasma hotspots include regions not occupied by nanoscale nucleic acid scaffolds, and (c) The nucleic acid polymerase binds to the nanoscale nucleic acid scaffold and is located in a region not occupied by the nanoscale nucleic acid scaffold, and (d) Optionally, nucleic acid analytes are bound to nanoscale nucleic acid scaffolds.

[0030] In the methods of this disclosure and / or other examples of plasmonic nanoantennas, the plasmonic nanoantenna may comprise more than two plasmonic nanoparticles, each bound to a nanoscale nucleic acid scaffold and positioned relative to each other such that plasmonic hot spots exist between the plasmonic nanoparticles. For example, a plasmonic nanoantenna may comprise three plasmonic nanoparticles. For example, a plasmonic nanoantenna may comprise four plasmonic nanoparticles. For example, a plasmonic nanoantenna may comprise five plasmonic nanoparticles. For example, a plasmonic nanoantenna may comprise six plasmonic nanoparticles. For example, a plasmonic nanoantenna may comprise seven plasmonic nanoparticles. For example, a plasmonic nanoantenna may comprise eight plasmonic nanoparticles. For example, a plasmonic nanoantenna may comprise nine plasmonic nanoparticles. For example, a plasmonic nanoantenna may comprise ten or more plasmonic nanoparticles.

[0031] According to the present disclosure, in which a plasma nanoantenna comprises two plasma nanoparticles and / or examples of a plasma nanoantenna, the region within the plasma hotspot not occupied by the nanoscale nucleic acid scaffold is defined by a three-dimensional space having a length (L) between the two plasma nanoparticles, a height (H) perpendicular to (L) at the midpoint (L / 2), and a width (W) perpendicular to both L and H at L / 2, wherein at L / 2, W is selected from about 20 nm to about 100 nm, and H is selected from about 10 nm to about 100 nm, and wherein L is measured at the shortest distance between the two plasma nanoparticles. In some examples, L is selected from about 20 nm to about 100 nm. In other examples, L is selected from about 20 nm to about 50 nm.

[0032] In other instances, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold is at least about 1 zL, such as about 1 zL to about 10 zL, or about 5 zL to about 10 zL. In some instances, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold is greater than or equal to about 10 zL. For example, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold is about 10 zL to about 50 zL. For example, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold can be about 20 zL to about 40 zL.

[0033] Based on the above examples, the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold is large enough to accommodate the nucleic acid polymerase and maintain its biological activity. In some examples, the region not occupied by the nanoscale nucleic acid scaffold is less than or equal to 50% of the plasma hotspot. However, in other examples, the region not occupied by the nanoscale nucleic acid scaffold is greater than 50% of the plasma hotspot. In some examples, the region not occupied by the nanoscale nucleic acid scaffold is 60% or more of the plasma hotspot. In some examples, the plasma hotspot is essentially not occupied by the nanoscale nucleic acid scaffold. For example, the region not occupied by the nanoscale nucleic acid scaffold can be 70% or more of the plasma hotspot. In some examples, the region not occupied by the nanoscale nucleic acid scaffold is 75% or more of the plasma hotspot. In some examples, the region not occupied by the nanoscale nucleic acid scaffold is 80% or more of the plasma hotspot. In some examples, the region not occupied by the nanoscale nucleic acid scaffold is 85% or more of the plasma hotspot. In some examples, the region not occupied by the nanoscale nucleic acid scaffold is 90% or more of the plasma hotspot.

[0034] In each of the above examples, the region not occupied by the nanoscale nucleic acid scaffold can exhibit different shapes or forms. In one example, the region not occupied by the nanoscale nucleic acid scaffold can be amorphous. Alternatively, the region not occupied by the nanoscale nucleic acid scaffold can have a substantially defined shape. For example, the region not occupied by the nanoscale nucleic acid scaffold can be spherical, elliptical, cubic, or cuboid.

[0035] According to the methods of this disclosure and / or some examples of plasma nanoantennas, the average particle spacing between plasma nanoparticles is from about 10 nm to about 100 nm, wherein the particle spacing between two particles is determined as the shortest distance between the respective particle surfaces. For example, the average particle spacing between plasma nanoparticles can be from about 10 nm to 80 nm. For example, the average particle spacing between plasma nanoparticles can be from about 10 nm to 50 nm. For example, the average particle spacing between plasma nanoparticles can be from about 20 nm to 80 nm. For example, the average particle spacing between plasma nanoparticles can be from about 20 nm to 50 nm. For example, the average particle spacing between plasma nanoparticles can be from about 30 nm to 50 nm. For example, the average particle spacing between plasma nanoparticles can be from about 20 nm to 40 nm. For example, the average particle spacing between plasma nanoparticles can be from about 30 nm to 40 nm. In one specific example, the average particle spacing between plasma nanoparticles is about 30 nm.

[0036] In some examples of the methods and / or plasma nanoantennas disclosed herein, the plasma nanoantennas comprise nucleic acid analytes bound to a nanoscale nucleic acid scaffold. For example, the nucleic acid analytes may be non-covalently bound (e.g., via hydrogen bonds) to the nanoscale nucleic acid scaffold.

[0037] The plasma nanoparticles of the plasma nanoantennas described herein can be formed from any material possessing plasma resonance properties. Suitable plasma nanoparticles include, but are not limited to, metallic nanoparticles, metallic alloy nanoparticles, polymeric nanoparticles and their derivatives, and composite materials. In one instance, each plasma nanoparticle is independently selected from metallic nanoparticles. For example, each of two or more plasma nanoparticles is an independently selected metallic nanoparticle, or an nanoparticle formed from an alloy of gold, silver, aluminum, copper, bismuth, nickel, palladium, and platinum nanoparticles. In some instances, the metallic nanoparticles are solid and formed entirely of metallic material. In other instances, the metallic nanoparticles are formed entirely of metallic material but have a hollow core. In still other instances, the metallic nanoparticles contain a core (e.g., a glass, polymer, or composite core) encased within a metallic material to form a metallic surface. In one instance, the plasma nanoparticle is a gold nanoparticle or a gold alloy nanoparticle. In one instance, the plasma nanoparticle is a silver nanoparticle or a silver alloy nanoparticle. In other instances, plasma nanoparticles are formed from nonmetallic materials or metal oxides that are doped to increase the total carrier load of the nanoparticles, thereby enhancing their plasma resonance.

[0038] The plasma nanoparticles in the plasma nanoantennas described herein can have different shapes and geometries. Therefore, the plasma nanoparticles can be independently selected from nanospheres, nanorods, nanoprisms, nanocubes, nanoshells, nanotubes, or nanostars. The plasma nanoparticles on the plasma nanoantenna can be of the same shape or they can be hybrid. In one example, the plasma nanoparticles are nanospheres.

[0039] The size of the plasma nanoparticles in the plasma nanoantennas described herein can vary. In one example, the maximum diameter of each plasma nanoparticle is about 5 nm to about 500 nm. In another example, the maximum diameter of each plasma nanoparticle is about 10 nm to about 250 nm. In another example, the maximum diameter of each plasma nanoparticle is about 20 nm to about 200 nm. In another example, the maximum diameter of each plasma nanoparticle is about 50 nm to about 150 nm. In another example, the maximum diameter of each plasma nanoparticle is about 80 nm to about 120 nm. In one specific example, the maximum diameter of each plasma nanoparticle is about 80 nm. In another specific example, the maximum diameter of each plasma nanoparticle is about 100 nm. In yet another specific example, the maximum diameter of each plasma nanoparticle is about 120 nm.

[0040] In some instances, the plasma nanoparticles have the same maximum diameter. In other instances, the maximum diameters of the plasma nanoparticles on the same plasma nanoantenna differ. Based on the instances where the maximum diameters of the plasma nanoparticles on the same plasma nanoantenna differ, the average maximum diameter of the plasma nanoparticles can be from about 5 nm to about 500 nm. For example, the average maximum diameter of the plasma nanoparticles on the plasma nanoantenna can be from about 10 nm to about 250 nm. For example, the average maximum diameter of the plasma nanoparticles on the plasma nanoantenna can be from about 20 nm to about 200 nm. For example, the average maximum diameter of the plasma nanoparticles on the plasma nanoantenna can be from about 50 nm to about 150 nm. For example, the average maximum diameter of the plasma nanoparticles on the plasma nanoantenna can be from about 80 nm to about 120 nm. In one specific instance, the average maximum diameter of the plasma nanoparticles on the plasma nanoantenna can be about 80 nm. In one specific instance, the average maximum diameter of the plasma nanoparticles on the plasma nanoantenna can be about 100 nm. In one specific instance, the average maximum diameter of the plasma nanoparticles on the plasma nanoantenna can be about 120 nm.

[0041] In one specific example, the plasma nanoantenna described herein comprises gold or gold-coated nanoparticles (e.g., nanospheres) with an average maximum diameter of about 100 nm.

[0042] In each of the above examples describing the plasma nanoantenna of this disclosure and / or its use in the methods of this disclosure, each plasma nanoparticle is coated with a coating layer. Preferably, the coating layer substantially covers the surface of each plasma nanoparticle. In one example, the coating layer covers about 75% or more of the surface of each plasma nanoparticle. In one example, the coating layer covers about 80% or more of the surface of each plasma nanoparticle. In one example, the coating layer covers about 85% or more of the surface of each plasma nanoparticle. In one example, the coating layer covers about 90% or more of the surface of each plasma nanoparticle. In one example, the coating layer covers about 95% or more of the surface of each plasma nanoparticle.

[0043] In some instances, the coating layer reduces the aggregation of plasma nanoparticles compared to uncoated plasma nanoparticles. In one instance, the coating layer reduces the aggregation of plasma nanoparticles by 35% or more compared to uncoated plasma nanoparticles. In another instance, the coating layer reduces the aggregation of plasma nanoparticles by 50% or more compared to uncoated plasma nanoparticles. In yet another instance, the coating layer reduces the aggregation of plasma nanoparticles by 65% ​​or more compared to uncoated plasma nanoparticles.

[0044] In each of the above examples describing the coating layer of plasma nanoparticles, the coating layer may comprise a plurality of oligonucleotides. In some examples, each nucleotide of each of the plurality of oligonucleotides is selected from pyrimidine nucleotides. In one example, each of the plurality of oligonucleotides comprises about 20 to about 50 nucleotides. In one example, each of the plurality of oligonucleotides comprises about 20 to about 30 nucleotides.

[0045] In each of the examples of the plasma nanoantennas described above and / or their use in the methods of this disclosure, the nanoscale nucleic acid scaffold can be substantially composed of double-stranded DNA. For example, the double-stranded DNA can be parallel interconnected double helical chains. In some examples, the nanoscale nucleic acid scaffold is composed of DNA origami structures (e.g., three-dimensional DNA origami structures). In one example, the DNA origami structure comprises M13mp18-derived scaffold chains and complementary staple chains. In some examples, at least a portion of the DNA origami structure is assembled to form a U-shaped or curved structure.

[0046] In one example, the nanoscale nucleic acid scaffold comprises a first face to which one of two plasmonic nanoparticles is anchored, and a second face to which the second of the two plasmonic nanoparticles is anchored. The first and second faces of the scaffold may be located on the same side or adjacent sides of the nanoscale nucleic acid scaffold. Alternatively, the first and second faces of the scaffold may be located on opposite sides of the nanoscale nucleic acid scaffold.

[0047] According to the present disclosure, a method and / or example of a plasma nanoantenna comprising two plasma nanoparticles is provided, wherein one plasma nanoparticle is anchored to a first surface of a scaffold via one or more nucleic acid connectors, and the other plasma nanoparticle is anchored to a second surface of the scaffold via one or more nucleic acid connectors. Each nucleic acid linker is formed by an oligonucleotide coated on the surface of one of the plasma nanoparticles, which hybridizes with a polynucleotide forming part of or extending from the nanoscale nucleic acid scaffold. The sequences of the corresponding oligonucleotides and polynucleotides capable of hybridization are complementary or substantially complementary to each other. According to this example, the nanoscale nucleic acid scaffold comprises one or more polynucleotides extending from a first side of the scaffold, each polynucleotide containing a sequence complementary to the sequence of one or more oligonucleotides coating one of the two plasma nanoparticles. Furthermore, the nanoscale nucleic acid scaffold comprises one or more polynucleotides extending from a second side of the scaffold, each polynucleotide containing a sequence complementary to the sequence of one or more oligonucleotides coating one of the two plasma nanoparticles. The polynucleotides forming part of or extending from the nanoscale nucleic acid scaffold can each be single-stranded DNA. Preferably, the polynucleotides extending from the first and second sides of the scaffold each contain a region of sufficient length and complementarity to hybridize with the oligonucleotides on the surface of the plasma nanoparticles. In one example, the polynucleotides extending from the first and second sides of the scaffold each contain about 20 to about 50 nucleotides. In another example, the polynucleotides extending from the first and second sides of the scaffold each contain about 20 to about 30 nucleotides. In each of these examples, the polynucleotides extending from the first and second sides of the scaffold each contain a region of about 10 to about 25 nucleotides in length, which is capable of hybridizing with a region of the corresponding length within the oligonucleotides on the surface of the plasma nanoparticles.

[0048] In each of the examples of the plasma nanoantennas described above and / or their use in the methods of this disclosure, a nucleic acid polymerase is bound to or immobilized on a nanoscale nucleic acid scaffold via a nucleic acid adapter. In one example, the nucleic acid adapter is double-stranded and comprises: (i) a polynucleotide covalently or non-covalently bound to the nucleic acid polymerase via amino acids within the nucleic acid polymerase; and (ii) a polynucleotide forming part of or extending from the nanoscale nucleic acid scaffold, wherein the polynucleotides at (i) and (ii) comprise complementary or substantially complementary single-stranded DNA sequences capable of hybridizing with each other. In one example, the polynucleotide bound to the nucleic acid polymerase comprises a peptide tag that binds to the nucleic acid polymerase via a heteropeptide bond formed between amino acids within the nucleic acid polymerase and amino acids within the peptide tag. In one example, the heteropeptide bond is capable of spontaneous formation. For example, the heteropeptide bond can be formed between lysine (Lys) and aspartic acid (Asp), or between lysine (Lys) and asparagine (Asn). In some examples, the amino acid within the nucleic acid polymerase bound to the nucleic acid adapter is located in the N-terminal domain of the nucleic acid polymerase.

[0049] Each polynucleotide of a nucleic acid linker, to which a nucleic acid polymerase is bound or immobilized on a nanoscale nucleic acid scaffold, contains a region of sufficient length and complementarity to hybridize with each other. In one example, each polynucleotide of the nucleic acid linker contains about 20 to about 50 nucleotides. In another example, each polynucleotide of the nucleic acid linker contains about 20 to about 30 nucleotides. In each of these examples, the polynucleotide of the nucleic acid linker each contains a region of about 10 to about 25 nucleotides in length that is complementary and capable of hybridizing with each other.

[0050] In each instance of the plasma nanoantenna described above and / or its use in the methods of this disclosure, the nucleic acid polymerase may be a DNA polymerase.

[0051] According to any of the examples described herein, where a nucleic acid polymerase (e.g., DNA polymerase) is bound to or immobilized on a nanoscale nucleic acid scaffold of a plasma nanoantenna via a nucleic acid adapter, the nucleic acid polymerase may be modified to express an amino acid sequence capable of binding to a peptide tag conjugated to the nucleic acid adapter.

[0052] This disclosure also provides an array comprising the plurality of plasma nanoantennas described herein.

[0053] In one example, the array comprises a solid substrate, and multiple plasmonic nanoantennas are fixed to this solid substrate. For example, each plasmonic nanoantenna in the array can be fixed to the solid substrate by its nucleic acid scaffold. In some examples, the solid substrate is glass or silica. For example, the solid substrate can be glass or silica in the form of a glass slide or a chip.

[0054] In some instances, less than 30% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas. For example, less than about 25% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas. For example, less than about 20% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas. For example, less than about 15% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas. For example, less than about 10% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas.

[0055] In some instances, the arrays disclosed herein can be used in the nucleic acid analyte sequencing methods described herein. Therefore, in any of the above-described examples of nucleic acid analyte sequencing methods, the arrays comprising multiple plasmonic nanoantennas described herein can be employed, for example, for simultaneously determining the sequences of multiple nucleic acid analytes.

[0056] Those skilled in the art will understand that various variations and / or modifications can be made to the above embodiments without departing from the broad scope of this disclosure. Therefore, the examples and embodiments herein should be considered illustrative rather than restrictive in all respects. Attached Figure Description

[0057] The following figures form part of this specification and are intended to further illustrate certain aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to one or more of these figures in conjunction with the detailed description of specific embodiments herein.

[0058] Figure 1 The design of the plasma nanoantenna DNA sequencer disclosed herein is illustrated.

[0059] Figure 2 The design of the NanoSpanno DNA origami scaffold disclosed herein is illustrated.

[0060] Figure 3The synthesis of the DNA origami NanoSpanno scaffold described herein is illustrated. (A) Agarose gel stained with RedSafe™ DNA staining solution. The gel ladder contains DNA molecular weight standards; lane 2, M13 contains the single-stranded M13 phage genome (7249 nt); lane 3, NS contains the synthesized NanoSpanno sample. (B) Typical transmission electron microscopy (TEM) image at 38,000x magnification, containing approximately 20 NanoSpanno particles. (C) Two-dimensional classification average plot of the image and a three-dimensional model showing the putative orientation.

[0061] Figure 4 Visualization results of thiol-coated DNA on gold nanoparticles are shown. (A) Agarose gel electrophoresis under white light transmission (top), where colloidal gold appears as a red band, and agarose gel electrophoresis after DNA-specific staining (bottom). (B) TEM micrographs of bare gold nanoparticles (left) and TEM micrographs of gold nanoparticles coated with thiol-coated DNA (right).

[0062] Figure 5 The stability of DNA-coated gold nanoparticles is illustrated. The DNA-coated gold nanoparticles were suspended in IxTAE + 6 mM MgCl2. Agarose gels were stained with DNA-specific dyes, and the images show the colors of the corresponding samples.

[0063] Figure 6 This diagram displays an expression vector map of recombinant DNA polymerases. The petDuet-1 vector is shown on the left, with relevant features labeled. The right side shows the MCS-1 site, which clones all three recombinant polymerases. The restriction sites and composition of each recombinant protein are shown in order from the N-terminus to the C-terminus.

[0064] Figure 7Example data are provided for IMAC and SEC purification of Klenow-SpyCatcher, as well as SEC purification of Klenow-SpyCatcher labeled with Alexa647. (A) shows the UV280 chromatogram of IMAC purification. The green line represents the sample pump gradient, and the orange line represents conductivity, providing a measure of the concentration of imidazole in the sample. (B) SDS-PAGE of the IMAC-purified eluent. (C) shows the UV280 chromatogram of SEC purification. (D) SDS-PAGE of the SEC-purified eluent. (E) shows the SEC purification chromatogram of Klenow-SpyCatcher labeled with Alexa647. The blue line represents the absorbance at 280 nm, and the purple line represents the absorbance at 650 nm. (F) SDS-PAGE of the SEC-purified eluent of Klenow-SpyCatcher labeled with Alexa647; the left gel is scanned with Alexa647, and the right gel is scanned under bright field.

[0065] Figure 8 The diagram illustrates the two-step process for synthesizing the DNA-SpyTag peptide. The left diagram depicts the reaction with SMCC to generate maleimide-DNA. The right diagram depicts the reaction of maleimide-DNA with the thiol cysteine ​​side chain of the peptide to generate a DNA-peptide conjugate.

[0066] Figure 9 Data related to the HPLC purification of maleimide DNA are presented. (A) HPLC chromatogram of 5'-maleimide-15nt DNA at 260 nm; (B) HPLC chromatogram of 3'-maleimide-15nt DNA; (C) Absorption spectrum of 5'Cy5-3'-maleimide-15nt DNA and absorbance at 260 nm (blue) and 650 nm (orange).

[0067] Figure 10 Data related to the HPLC chromatography of peptide-DNA conjugates are presented. (A) HPLC chromatogram of 5'-SpyTag-15nt DNA at 260 nm. (B) HPLC chromatogram of 3'-SpyTag-15nt DNA at 260 nm. (C) Absorption spectrum of 5'Cy5-3'-SpyTag-15nt DNA and absorbance at 260 nm (blue) and 650 nm (orange).

[0068] Figure 11SDS-PAGE gel images showing the conjugation of SpyTag-DNA to polymerase-SpyCatcher are displayed. (A) The left image shows Alexa 647 imaging, while the right image shows a bright-field image of the gel after protein-specific staining. At the top, the 5'-SpyTag-DNA conjugate binds to the Alexa 647-labeled Klenow-SpyCatcher, and at the bottom, the 3'-SpyTag-DNA conjugate binds to the Alexa 647-labeled Klenow-SpyCatcher. In each image, the left lane shows the Alexa 647-labeled Klenow, and the right lane shows the Alexa 647-labeled Klenow conjugated with SpyTag-DNA. (B) The gel shows the 3'-SpyTag-DNA-5'Cy5 conjugate bound to the unlabeled Klenow-SpyCatcher. The left image shows the merged imaging result, the middle image shows the Cy5 imaging result, and the right image shows a bright-field image of the gel after protein-specific staining. In each image, the left lane shows the Klenow-SpyTag-DNA conjugate, and the right lane shows Klenow.

[0069] Figure 12 The functions of DNA-tagged polymerases are illustrated. (A) 15% Tris-glycine natural gel used for DNA polymerase (Klenow, Tag, Phi29) activity assay. (B) 15% Tris-glycine natural gel used for Klenow-SpyCatcher-SpyTag-DNA conjugation (left: Alexa488, right: after SYBR™ Gold nucleic acid gel staining).

[0070] Figure 13 The binding kinetics of Klenow (top row) and Phi29 (bottom row) to the primer-bearing target DNA strand are shown. The binding curves (A, D) and dissociation curves (B, E) are both fitted as biphasic exponential curves (red), with the fitting conditions described in the figure for a range of protein concentrations. The plateau phases of the binding curves are plotted against protein concentrations (C, F) and fitted as a unit point binding model (red).

[0071] Figure 14 The assembly of the plasma nanoantenna of this disclosure is shown. (A) i: Image showing the solution colors; ii: Agarose gel electrophoresis image where the red bands correspond to colloidal gold; iii: Agarose gel electrophoresis image after DNA-specific staining. (B) Example images of well-structured dimers with different sequences observed by electron microscopy and cryo-electron microscopy. (C) Example images of monomers and aggregates.

[0072] Figure 15The yields for different configurations and sequences were plotted: (A) 100 nm AuNP-25AC + NS-25TG (2 binding sites); (B) 100 nm AuNP-25AC + NS-25TG (1 binding site); (C) 100 nm AuNP-25AC + NS-25TG (0 binding sites); (D) 100 nm AuNP-25TG + NS-25AC (2 binding sites); (E) 100 nm AuNP-25TG + NS-25AC (1 binding site); (F) 100 nm AuNP-25TG + NS-25AC (0 binding sites).

[0073] Figure 16 The illustration shows DNA polymerase binding to the cavity of a DNA origami NanoSpanno scaffold via DNA hybridization.

[0074] Figure 17 The specific co-localization of DNA polymerase with the DNA origami scaffold was demonstrated. 2% agarose gel was used to confirm the DNA origami scaffold and Klenow conjugation, and 2% agarose gel was used as a negative control.

[0075] Figure 18 The colocalization yield of DNA polymerase with the DNA origami scaffold is shown. Klenow display data are presented for the DNA origami scaffold labeled with Alexa488 and Alexa647. (A) shows an example fluorescence image with intensity traces of the circled region shown in (B). (C) shows the distribution of initial Alexa-647 and initial Alexa-488 intensities. (D) is the experimentally determined colocalization yield of DNA polymerase binding to different locations in the cavity of the DNA origami scaffold, as depicted in the inset image. NStop corresponds to the DNA origami annealed with a molar excess of Alexa647 DNA, which is complementary to the staple extension at the edge of the cavity.

[0076] Figure 19The following shows the quantification of fluorescence enhancement in the presence of AuNP. (A) Exemplary photobleaching trace as shown in the figure. (B) Photobleaching step height distribution used to measure the intensity of a single Alexa647 fluorophore on a DNA origami scaffold in the absence of AuNP binding sites (blue) and in the presence of AuNP binding sites (red). (C) Quantification of photobleaching rate based on the data shown in (B). (D) Scattering distribution from 2xAuNP binding sites measured from channels 488 and 647, with threshold selection using Gaussian fitting of channel 488. (E) Step height distribution of particles in 2xAuNP experiments based on scattering from channel 488 as shown in (D). (F) Photobleaching rate based on the data shown in the figure.

[0077] Figure 20 The results show the results of repeated measurements of fluorophore intensity in hotspot regions by sequentially binding and dissociating short Alexa647-tagged DNA strands (imager strands) in the absence of 2AuNP (top image) or in the presence of 2AuNP (bottom image).

[0078] Figure 21 The results show the repetitive intensity measurements of the plasmonic nanoantenna in the absence (orange) and presence (blue) of the 1 nM imaging chain.

[0079] Figure 22 The signal amplification of a single fluorescent DNA polymerase bound to a hotspot is shown.

[0080] Figure 23 The scattering intensity of vertically aligned plasmonic nanoantennas varies with polarization. (A) i. Simulated TIRF background intensity as the incident light polarization angle increases; ii. Experimentally measured background intensity as the polarization angle increases. (B) i. Predicted scattering cross-section of dimer AuNPs aligned along the TIRF field propagation axis (black) or perpendicular to the TIRF field propagation axis (blue); ii. Measured particle intensity as the polarization angle increases, from which the particle number and orientation are inferred. Dimerer AuNPs aligned along the TIRF field propagation axis (black) or perpendicular to the TIRF field propagation axis (blue), or monomeric AuNPs (green).

[0081] Figure 24 Traces of a single plasmonic nanoantenna are shown after incubation with biotin-DNA complementary to AuNP-bound DNA, in the absence of an imaging strand. Figure 21 and Figure 22 In comparison, the volatility was significantly reduced.

[0082] Figure 25 The illustration shows the simulated enhancement of the Alexa647 fluorophore located within the hot spot under different orientations of the plasma nanoantenna relative to the polarization of the incident light.

[0083] Figure 26 Fluorescent labeling of free dNTP molecules is shown, including HPLC chromatograms of purified fluorescently labeled dNTPs.

[0084] Figure 27 The configuration of the plasma nanoantenna DNA sequencer of this disclosure for single-molecule DNA sequencing reactions, as described herein, is shown.

[0085] Figure 28 Example traces from a two-color DNA sequencing experiment are shown. These traces are derived from two separate plasma nanoantenna DNA sequencer molecules. The sequencing reaction was performed using Alexa647-labeled dGTP and Alexa561-labeled dATP, with dCTP and dTTP nucleotides unlabeled. The traces indicate bases identified by intensity values ​​three standard deviations above the background. Detailed Implementation

[0086] Overview Throughout this specification, unless otherwise expressly stated or required by context, reference to a single step, feature, composition of substance, group of steps, or group of features or composition of substance shall include one or more (i.e., one or more) of such steps, features, compositions of substance, groups of steps, or groups of features or composition of substance.

[0087] Those skilled in the art will understand that variations and modifications can be made to this disclosure beyond those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds mentioned or specified in this specification, whether individually or collectively, and any and all combinations of any two or more of said steps or features.

[0088] The scope of this disclosure should not be limited to the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are obviously within the scope of this disclosure.

[0089] Unless otherwise expressly stated, any instance in this disclosure shall be regarded as any other instance applicable to this disclosure after necessary modifications in detail.

[0090] Unless otherwise expressly defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0091] Unless otherwise stated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. These techniques are described and explained in the following literature: 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); TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 & 2, IRL Press (1991); DM Glover and BD Hames (ed.), DNA Cloning: A Practical Approach, Volumes 1–4, IRL Press (1995 and 1996); and FM Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (ed.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); and JE Coligan et al. (ed.), Current Protocols. inImmunology, John Wiley & Sons (including all updates to date).

[0092] Those skilled in the art will understand that variations and modifications can be made to this disclosure beyond those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds mentioned or specified in this specification, whether individually or collectively, and any and all combinations of any two or more of said steps or features.

[0093] The scope of this disclosure should not be limited to the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are obviously within the scope of this disclosure, as described herein.

[0094] Each feature of any particular aspect or implementation of this disclosure may be applied to any other aspect or implementation of this disclosure after necessary modifications to the details.

[0095] Throughout this specification, unless otherwise expressly stated or required by context, references to a single step, composition of substance, group of steps, or group of composition of substance shall include one or more (i.e., one or more) of such steps, compositions of substance, groups of steps, or groups of composition of substance.

[0096] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations thereof, such as “comprises” or “comprising”, shall be understood to imply inclusion of the stated steps or elements or integers or groups of steps or elements or integers, but not to exclude any other steps or elements or integers or groups of elements or integers.

[0097] The term “and / or”, for example, “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be regarded as explicitly supporting either or both of these meanings.

[0098] Plasma nanoantenna This invention discloses a plasma nanoantenna comprising: (i) Two plasma nanoparticles; (ii) Nanoscale nucleic acid scaffolds; and (iii) Nucleic acid polymerase; in: (a) Two plasma nanoparticles bind to a nanoscale nucleic acid scaffold and are positioned relative to each other, resulting in plasma hotspots between the plasma nanoparticles. (b) Plasma hotspots include regions not occupied by nanoscale nucleic acid scaffolds. (c) The nucleic acid polymerase binds to the nanoscale nucleic acid scaffold and is located in a region (plasma hotspot) not occupied by the nanoscale nucleic acid scaffold, and Optionally, nucleic acid analytes are bound to nanoscale nucleic acid scaffolds.

[0099] Figure 1 The illustration shows a plasma nanoantenna according to an exemplary embodiment of the present disclosure.

[0100] As used herein, the term "plasma nanoparticle" refers to particles with nanoscale dimensions whose electron density can couple with electromagnetic radiation with wavelengths much larger than the particles themselves due to the properties of the dielectric-metal interface between the medium and the particles. It should be understood that plasma nanoparticles can exhibit different scattering, absorption, and coupling properties depending on their geometry and relative position; different geometries are considered herein. The nanoparticles disclosed herein can be formed from any material with plasmon resonance properties. Suitable nanoparticles include, but are not limited to, metallic nanoparticles, metallic alloy nanoparticles, polymeric nanoparticles and their derivatives, and composite materials. In one specific example, each plasma nanoparticle is independently selected from metallic nanoparticles.

[0101] As used herein, the term "metal nanoparticle" should be understood to mean particles of nanoscale size composed of metallic substances. It should be understood that nanoparticles can be formed into any suitable or desired shape and size, examples of which will be described herein. Metal nanoparticles can be formed wholly or partially from any metallic material with plasmon resonance properties. The metallic nanoparticles of the plasmon nanoantennas described herein can be formed wholly or partially from any metallic material with plasmon resonance properties known in the art. Known metallic materials with plasmon resonance properties include noble metals and non-noble metals. Suitable examples include, but are not limited to, gold, silver, aluminum, copper, bismuth, nickel, palladium, and platinum nanoparticles or alloys of any of them. For example, one or both or each of the metallic nanoparticles may be gold nanoparticles, gold alloy nanoparticles, or nanoparticles with a gold or gold alloy coating. For example, one or both or each of the nanoparticles may be silver nanoparticles, silver alloy nanoparticles, or nanoparticles with a silver or silver alloy coating. For example, one or both or each of the nanoparticles may be aluminum nanoparticles, aluminum alloy nanoparticles, or nanoparticles with an aluminum or aluminum alloy coating. For example, one or both of the nanoparticles may be copper nanoparticles, copper alloy nanoparticles, or nanoparticles with a copper or copper alloy coating. For example, one or both of the nanoparticles may be bismuth nanoparticles, bismuth alloy nanoparticles, or nanoparticles with a bismuth or bismuth alloy coating. For example, one or both of the nanoparticles may be nickel nanoparticles, nickel alloy nanoparticles, or nanoparticles with a nickel or nickel alloy coating. For example, one or both of the nanoparticles may be palladium nanoparticles, palladium alloy nanoparticles, or nanoparticles with a palladium or palladium alloy coating. For example, one or both of the nanoparticles may be platinum nanoparticles, platinum alloy nanoparticles, or nanoparticles with a platinum or platinum alloy coating.

[0102] Based on examples of plasma nanoparticles comprising a core encapsulated with a metallic material having plasma resonance properties as described herein, the core may be formed of glass, polymer or composite core or other suitable material.

[0103] Nanoparticles formed from nonmetallic materials and metal oxides can also be considered as plasma nanoparticles. For example, one or more of the plasma nanoparticles may be formed from nonmetallic materials or metal oxides doped to have or enhance plasma resonance properties. Exemplary nonmetallic materials include, but are not limited to, metal oxides, chalcogenides, phosphides, nitrides, and silicon. Doped plasma nanoparticles and methods for their preparation have been described in the literature and are known to those skilled in the art, including, but not limited to, optical doping, chemical doping, hierarchical doping, and combinations thereof.

[0104] Therefore, in one instance, one or more plasmonic nanoparticles are formed from photo-doped nonmetallic materials or metal oxides. The method of using photo-doped nanoparticles to impart or enhance plasmonic resonance properties is described in Petrini et al., (2023). J. Phys. Chem. C. Nanomater Interfaces In , 127(3): 1576-1587. In another example, one or more plasmonic nanoparticles are formed from hierarchically doped nonmetallic materials or metal oxides. The method of hierarchically doped nanoparticles that can be used to impart or enhance plasmonic resonance properties is described in Kim et al., (2023). Nano Letters , 23(16):7633-7641 and Russo et al., (2018) Materials & Designs In another example, one or more plasmonic nanoparticles are formed from chemically doped nonmetallic materials or metal oxides, for example, by introducing metal atoms into the host lattice or structure. Examples of materials that can be chemically doped to improve carrier density and plasmonic resonance properties include metal oxides, chalcogenides, phosphides, nitrides, and silicon. Methods for chemically doped nanoparticles that can be used to impart or enhance plasmonic resonance properties are described herein in Chowdhury et al., (2017). Nanoscale , 9: 15591-15597 and Liu et al., (2019) Nature Communications , 10: 1394 in.

[0105] In some instances, the plasma nanoparticles are formed of or contain the same material. For example, each plasma nanoparticle may be a metallic plasma nanoparticle as described herein. For example, each plasma nanoparticle may be a gold nanoparticle, or a nanoparticle coated with gold to have a gold surface. For example, each plasma nanoparticle may be a silver nanoparticle, or a nanoparticle coated with silver to have a silver surface.

[0106] In other instances, the plasmonic nanoparticles are formed of or contain materials that are different from each other. For example, one nanoparticle may be a gold nanoparticle or a gold-coated nanoparticle, while one or more other nanoparticles may be formed of or coated with another material (such as another noble metal (such as silver) or other metals that have plasmonic resonance properties as described herein).

[0107] The plasma nanoparticles described herein can be formed into any suitable or desired shape and size, such as nanospheres, nanorods, nanoprisms, nanocubes, nanoshells, nanotubes, or nanostars. It should be understood that these are merely representative examples of nanoparticle shapes and do not limit the scope of this disclosure in any way. In one example, one or more plasma nanoparticles, or each plasma nanoparticle, is a nanosphere. In one example, one or more plasma nanoparticles, or each plasma nanoparticle, is a nanorod. In one example, one or more plasma nanoparticles, or each plasma nanoparticle, is a nanoprism. In one example, one or more plasma nanoparticles, or each plasma nanoparticle, is a nanocube. In one example, one or more plasma nanoparticles, or each plasma nanoparticle, is a nanoshell. In one example, one or more plasma nanoparticles, or each plasma nanoparticle, is a nanotube. In one example, one or more plasma nanoparticles, or each plasma nanoparticle, is a nanostar. The plasma nanoparticles on the plasma nanoantenna can be of the same shape, or they can be hybrid.

[0108] In addition to shape, those skilled in the art will understand that the size of the nanoparticles in a plasma nanoantenna may vary. In some instances, the maximum diameter of the plasma nanoparticles may be the same or substantially the same. For example, the maximum diameter of each plasma nanoparticle may range from about 5 nm to about 500 nm (e.g., from about 10 nm to about 250 nm, or from about 20 nm to about 200 nm, or from about 50 nm to about 150 nm, or from about 80 nm to about 120 nm). In some instances, the maximum diameter of each plasma nanoparticle is about 80 nm. In some instances, the maximum diameter of each plasma nanoparticle is about 100 nm. In some instances, the maximum diameter of each plasma nanoparticle is about 120 nm.

[0109] In other instances, the maximum diameter of the plasma nanoparticles on the same plasma nanoantenna may differ. Based on examples where the maximum diameter of the plasma nanoparticles on the same plasma nanoantenna differs, the average maximum diameter of the plasma nanoparticles can range from about 5 nm to about 500 nm. For example, the average maximum diameter of the plasma nanoparticles on the plasma nanoantenna can range from about 10 nm to about 250 nm (e.g., within the ranges of about 20 nm to about 200 nm, 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 specific example, the plasma nanoparticles are gold or gold-coated nanoparticles (e.g., nanospheres) with an average diameter of about 100 nm. In another example, the plasma nanoparticles are silver or silver-coated nanoparticles (e.g., nanospheres) with an average diameter of about 100 nm.

[0110] As described herein, the plasma nanoantenna of this disclosure comprises two plasma nanoparticles, each attached to a nanoscale nucleic acid scaffold and positioned relative to each other, resulting in plasma hot spots between the plasma nanoparticles. However, in some instances, the plasma nanoantenna may comprise more than two plasma nanoparticles (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more nanoparticles), each attached to a nanoscale nucleic acid scaffold and positioned relative to each other, resulting in shared plasma hot spots between the plasma nanoparticles. Based on the instances where the plasma nanoantenna may comprise more than two plasma nanoparticles, the two or more plasma nanoparticles may aggregate together.

[0111] This document uses the terms "plasma hotspot," "plasma-enhanced hotspot," or similar terms to define a region between two or more plasma nanoparticles where the electric field is enhanced relative to the electric field outside the defined region between the two or more plasma nanoparticles. In one instance, a "plasma hotspot" refers to a region of enhanced electric field due to plasma resonance (i.e., polarization of plasma nanoparticles induced by an incident oscillating electric field, such as light).

[0112] Plasma nanoparticles are attached to a nanoscale nucleic acid scaffold and positioned relative to each other, resulting in plasma hotspots between the plasma nanoparticles. These hotspots contain regions not occupied by the nanoscale nucleic acid scaffold. As used herein, the phrase "regions not occupied by the nanoscale nucleic acid scaffold" or similar phrases should be understood as referring to the area or space within the plasma hotspot that a nucleic acid polymerase can enter (e.g., when it binds to the nanoscale nucleic acid scaffold). This unoccupied area may also be referred to as "vacant space" or "vacant hotspot" within the plasma hotspot. Preferably, the "regions not occupied by the nanoscale nucleic acid scaffold" are large enough that the polymerase's enzymatic activity is maintained when the nucleic acid polymerase is located within this area. In other words, the polymerase's enzymatic activity is not lost due to the steric hindrance effect (e.g., blockage) of the nanoscale nucleic acid scaffold. Methods for measuring and / or determining the size and / or volume of plasma hotspots between at least two plasma particles and vacant spaces accessible to the analyte are known in the art, for example, as Close et al., (2022). Advanced Materials Interfaces As stated in 9(24), and with particular reference to the Supplement. Figure 12(S12). For example, for the plasma nanoantenna described herein, if its vacant space is a cube with dimensions L = W = H = 30 nm, the vacant volume of the hotspot can be calculated as 2.7e4 nm^3 = 2.7e-20 L = approximately 27 zL, where 1 nm^3 equals 1e-24 L and 1 zL equals 1e-21 L. For the plasma nanoantenna described herein, if its vacant space is a sphere with a radius (r) of 15 nm, its vacant volume can be calculated as 4 / 3πr^3 = approximately 14 zL, where 1 nm^3 equals 1e-24 L and 1 zL equals 1e-21 L. Those skilled in the art should also understand that the size and / or volume of the region not occupied by the nanoscale nucleic acid scaffold can be determined based on the volume of the nanoscale nucleic acid scaffold that overlaps with the volume and / or size of the plasma hotspot. When designing hotspots of sufficient size and strength, those skilled in the art may also consider the following factors, including the size and design of the nanoscale nucleic acid scaffold, the wavelength of the incident light, the polarization of the incident light, the propagation of the incident light, the material of the nanoparticles, the shape of the nanoparticles, the volume of the nanoparticles, the spacing between the particles, the number of nanoparticles, the relative position of the nanoparticles, the dielectric constant of the surrounding medium, and / or the wavelength of the emission detection.

[0113] In some instances, the region within the plasma hotspot not occupied by the nanoscale nucleic acid scaffold is defined by a three-dimensional space having a length (L) between two plasma nanoparticles, a height (H) perpendicular to (L) at the midpoint (L / 2), and a width (W) perpendicular to both L and H at L / 2, 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 plasma nanoparticles. Those skilled in the art will understand that the shape of the nanoparticles (examples of which are described herein) and the geometry of the nucleic acid scaffold can affect the height and width of the plasma hotspot, as well as the height (H) and width (W) of the unoccupied region within the plasma hotspot. Those skilled in the art will also understand that when plasma nanoparticles are attached to a plasma nanoantenna, the particle spacing can be altered (or “tuned”) to change the size and intensity of the plasma hotspot, as well as the region unoccupied by the nanoscale nucleic acid scaffold. As used herein, “particle spacing” refers to the distance between any two plasma nanoparticles on the nanoscale DNA scaffold of the plasma nanoantenna. In some instances, the particle spacing L between the plasma nanoparticles is from about 10 nm to about 100 nm (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 about 80 nm). In another example, the interparticle spacing L between the plasma nanoparticles 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 interparticle spacing L of the plasma nanoparticles is about 20 nm to about 50 nm. In a particular example, the interparticle spacing L of the plasma nanoparticles is about 30 nm. In some examples, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold is at least about 1 zL, such as about 1 zL to about 10 zL, or about 5 zL to about 10 zL. In some examples, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold is greater than or equal to about 10 zL.In some instances, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold is at least about 10 zL (e.g., at least about 15 zL, or at least about 20 zL, or at least about 25 zL, or at least about 30 zL, or at least about 35 zL, or at least about 40 zL, or at least about 50 zL). For example, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold can be from about 10 zL to 50 zL (e.g., selected from about 10 zL to about 50 zL, or about 20 zL to about 50 zL, or about 30 zL to about 50 zL, or about 40 zL to about 50 zL, or about 10 zL to about 40 zL, or about 20 zL to about 40 zL, or about 30 zL to about 40 zL, or about 10 zL to about 30 zL, or about 20 zL to about 30 zL, or about 40 zL to about 50 zL). In one instance, the volume of the plasma hotspot region not occupied by the nanoscale nucleic acid scaffold was approximately 20 zL to approximately 40 zL.

[0114] Alternatively or additionally, the plasma hotspot area not occupied by the nanoscale nucleic acid scaffold can be expressed as a relative percentage of the total plasma hotspot volume. In some instances, the area not occupied by the nanoscale nucleic acid scaffold is 70% or more of the plasma hotspot (e.g., 75% or 80% or 85% or 90% or 95% or more).

[0115] Those skilled in the art will understand that plasma hotspots and plasma hotspot regions not occupied by the nanoscale nucleic acid scaffold can exhibit different shapes or forms, depending, for example, on the choice of nanoparticles and the geometry of the nucleic acid scaffold. In one instance, the region not occupied by the nanoscale nucleic acid scaffold can be amorphous. Alternatively, the region not occupied by the nanoscale nucleic acid scaffold can have a substantially defined shape. For example, the region not occupied by the nanoscale nucleic acid scaffold can be spherical, elliptical, cubic, or cuboid.

[0116] Plasma nanoparticles can be immobilized at predetermined locations on the surface of a nanoscale nucleic acid scaffold by: (i) DNA (i.e., oligonucleotides) coating the plasma nanoparticles; and (ii) DNA (i.e., polynucleotides) located at predetermined locations on the surface of the nanoscale nucleic acid scaffold and extending from the surface of the nanoscale nucleic acid scaffold, which are complementary or substantially complementary to the DNA coating the plasma nanoparticles (i.e., the nanoparticles can be covalently immobilized by Watson-Crick base pairing). In this way, the interparticle spacing of the plasma nanoparticles on the nanoscale nucleic acid surface can be controlled by rationally designing and positioning the polynucleotides extending from the surface of the nanoscale nucleic acid scaffold.

[0117] In some instances, plasma nanoparticles may be coated with or conjugated / linked to multiple oligonucleotides capable of hybridizing with polynucleotides extending from the surface of a nanoscale nucleic acid scaffold. For example, each plasma nanoparticle may be coated with multiple DNA oligonucleotides selected from 2 to about 100 oligonucleotides (e.g., 2 to about 75 oligonucleotides, or 2 to about 50 oligonucleotides, or 2 to about 25 oligonucleotides, or 2 to about 10 oligonucleotides). In other instances, each plasma nanoparticle may be coated with 5 or more DNA oligonucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more DNA oligonucleotides). Preferably, the DNA oligonucleotide coating layer substantially covers the surface of each plasma nanoparticle. For example, the DNA oligonucleotide coating layer may cover about 75% or more of the surface of each plasma nanoparticle (e.g., selected from about 80%, about 85%, about 90%, or about 95% or more).

[0118] In addition to promoting the immobilization of coated plasma nanoparticles on the surface of nanoscale nucleic acid scaffolds, during the fabrication of plasma nanoantennas, DNA oligonucleotides coated on plasma nanoparticles also reduce the aggregation of plasma nanoparticles compared to those without a coating layer. Exemplary methods for measuring nanoparticle aggregation are known in the art, including but not limited to ultraviolet-visible spectroscopy, differential centrifugation, particle counting and / or 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 In 35(14):4927-4935. In one instance, the DNA oligonucleotide coating layer reduced the aggregation of plasma nanoparticles by 35% or more (e.g., 40% or 45% or more) compared to uncoated plasma nanoparticles. In one instance, the DNA oligonucleotide coating layer reduced the aggregation of plasma nanoparticles by 50% or more (e.g., 55% or 60% or more) compared to uncoated plasma nanoparticles. In one instance, the DNA oligonucleotide coating layer reduced the aggregation of plasma nanoparticles by 65% ​​or more (e.g., 70% or 75% or more) compared to uncoated plasma nanoparticles.

[0119] The coating or conjugation of one or more DNA molecules (e.g., oligonucleotides) to plasma nanoparticles can be performed using any method known in the art. However, in one instance, one or more oligonucleotides bind to the plasma nanoparticles via a linker. In a specific instance, the oligonucleotides may be thiolized according to the embodiments disclosed herein. However, it should be understood that other functional anchoring groups, such as -COOH, -NH2, and -OH, may also be used to covalently attach DNA oligonucleotides to the surface of the nanoparticles.

[0120] The DNA oligonucleotides coated with plasma-encapsulated nanoparticles are designed such that their nucleotide sequences are complementary or substantially complementary to homologous DNA polynucleotide sequences extending from the surface of the nanoscale nucleic acid scaffold, enabling hybridization between the two. In some instances, more than 50% (e.g., more than 50%, 60%, 70%, or 80%) of the oligonucleotides or multiple oligonucleotides coated with plasma-encapsulated nanoparticles are selected from pyrimidine nucleotides (i.e., thymine and cytosine). In some instances, each nucleotide in the oligonucleotides or multiple oligonucleotides coated with plasma-encapsulated nanoparticles is selected from pyrimidine nucleotides (i.e., thymine and cytosine).

[0121] DNA oligonucleotides attached to plasma nanoparticles and complementary or substantially complementary DNA polynucleotides located within or extending from nanoscale nucleic acid scaffolds can be modified or unmodified. Suitable modifications are known in the art. These include, for example, azo modifications known in the art, which can be used to achieve photoresponsiveness and reversible binding and dissociation of the corresponding DNA molecules. Other modifications include modifications that modulate the strength of interactions between the corresponding DNA molecules, such as scaffold modifications that alter surface charge (e.g., peptide nucleic acids (PNAs)) or structural flexibility to induce hybridization efficiency (e.g., locked nucleic acids (LNAs)). Further modifications include the use of photolytically cleavable structures known in the art.

[0122] As described herein, plasma nanoantennas comprise nanoscale nucleic acid scaffolds to which plasma nanoparticles and nucleic acid polymerase molecules are immobilized or bonded. The nanoscale nucleic acid scaffolds can be nanoscale DNA scaffolds. Nanoscale DNA scaffolds can comprise one or more DNA origami structures or be formed from one or more DNA origami structures. The term “DNA origami structure” and similar terms should be understood to refer to a scaffold or structure formed from DNA molecules that self-assemble and are prepared using so-called DNA origami techniques. Scaffolded DNA origami is a technique proposed by Paul Rothemund in 2006, building upon earlier structural DNA nanotechnology and providing a pathway to obtaining finite DNA nanoobjects. In this technique, single-stranded circular DNA molecules, typically 7.25 kb in size, are folded into a helical array by periodic cross-alignment using staple chains. Extending this concept to assembling three-dimensional DNA structures has established a robust and reliable method for preparing DNA nanostructures (or “nanoscale DNA scaffolds”) in the size range of 20 to 100 nm. However, larger structures are also possible. DNA origami structures can be tubular or tubular, such as 3-helix, 6-helix, or 12-helix bundles, or can have a generally flat rectangular shape. In this way, DNA origami structures of different sizes and dimensions can be combined (e.g., stacked or layered) to construct nanoscale DNA scaffolds of arbitrary specific shapes. Origami structures can serve as “molecular staples” with addressable surface areas reaching thousands of nm. 2 This is used to align arbitrary target objects (OOIs) at a “single-pixel” resolution of approximately 6 nm. Furthermore, the shape, size, and contour of the origami structure surface can be customized to give the nanoscale DNA scaffold a surface geometrically suitable for attaching target molecules (e.g., the plasma nanoparticles and polymerases described herein). Methods for generating DNA origami structures (including those with DNA sequence strands protruding from the structure) are known in the art. In one instance, the nanoscale DNA scaffold comprises one or more three-dimensional DNA origami structures. In some instances, at least a portion of the DNA origami structure is assembled into a U-shape or curved structure. An exemplary U-shaped structure is shown below. Figure 1-3 As shown in the diagram.

[0123] Based on the example of assembling a U-shaped nanoscale DNA scaffold, the nanoscale DNA scaffold will include a first side and a second side. The first side anchors one of two plasma nanoparticles via a nucleic acid adapter, and the second side anchors the second of the two plasma nanoparticles via a nucleic acid adapter. The first and second sides of the scaffold can be located on the same side or adjacent sides of the nanoscale DNA scaffold, or on opposite sides thereof.

[0124] In one example, the nanoscale DNA scaffold will comprise: (i) one or more DNA polynucleotides extending from a first side of the scaffold, each polynucleotide containing a sequence complementary to the sequence of one or more DNA oligonucleotides coating one of the two plasma nanoparticles; and (ii) one or more DNA polynucleotides extending from a second side of the scaffold, each polynucleotide containing a sequence complementary to the sequence of one or more DNA oligonucleotides coating one of the two plasma nanoparticles. The DNA polynucleotides forming part of or extending from the nanoscale DNA scaffold may each be single-stranded DNA or contain single-stranded DNA. The DNA polynucleotides extending from the first and second sides of the scaffold will each contain a region of single-stranded DNA with sufficient length and complementarity to the DNA oligonucleotides coating the plasma nanoparticles, thereby enabling hybridization. Once homologous DNA polynucleotides hybridize with DNA oligonucleotides to form nucleic acid adapters, the plasma nanoparticles containing DNA oligonucleotides are anchored to the surface of the nanoscale DNA scaffold. Each nucleic acid adapter anchoring the plasma nanoparticle to the nanoscale DNA scaffold will be formed by hybridization of one of the DNA oligonucleotides coating the surface of the plasma nanoparticle with a DNA polynucleotide forming part of or extending from the nanoscale DNA scaffold. In one instance, the DNA polynucleotides extending from the first and second sides of the scaffold contain an M13mp18-derived scaffold sequence, and the DNA oligonucleotides encapsulated in the plasma nanoparticles contain a staple sequence complementary to it.

[0125] The length of the DNA polynucleotides extending from the first and second sides of the scaffold can be varied as needed. However, in some instances, the DNA polynucleotides extending from the first and second sides of the scaffold each contain about 20 to about 50 nucleotides (e.g., about 20 to about 40 nucleotides, or about 20 to about 30 nucleotides, or about 30 to about 50 nucleotides, or about 30 to about 50 nucleotides). In one instance, the DNA polynucleotides extending from the first and second sides of the scaffold contain about 20 to about 30 nucleotides.

[0126] In some instances, each plasma nanoparticle is anchored to a nanoscale DNA scaffold via multiple nucleic acid adapters (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). It is understood that the number of nucleic acid adapters connecting each plasma nanoparticle is positively correlated with the binding strength between the plasma nanoparticle and the nanoscale DNA scaffold. Therefore, the number of DNA polynucleotides extending from the nanoscale DNA scaffold and hybridizing with the DNA oligonucleotides coated on the plasma nanoparticles can be freely selected, depending on the specific target application, to achieve the desired binding strength and / or hybridization efficiency. Furthermore, the length of the Watson-Crick pairing region (i.e., the region between the DNA polynucleotide extending from the nanoscale DNA scaffold and the DNA oligonucleotide attached to the plasma nanoparticle) in the nucleic acid adapters can be varied, depending on the specific target application, to achieve the desired binding specificity and / or binding strength and / or hybridization efficiency. In some instances, the length of the Watson-Crick pairing region is approximately 8-30 nucleotides, or approximately 10-25 nucleotides, or approximately 12-20 nucleotides. As used in this article, the terms “hybridise,” “hybridising,” or similar terms refer to the process by which two complementary or substantially complementary nucleic acid sequences anneal each other according to the Watson-Crick base pairing rule.

[0127] Those skilled in the art will understand that the DNA oligonucleotides attached to the plasma nanoparticles, and the complementary or substantially complementary DNA polynucleotides located within or extending from the nanoscale DNA scaffold (which together hybridize to form nucleic acid adapters, anchoring the plasma nanoparticles to the nanoscale DNA scaffold), can be of arbitrary length. For example, the corresponding DNA molecule length can be independently selected from about 20 to about 50 nucleotides, about 20 to about 40 nucleotides, or about 20 to about 30 nucleotides. Preferably, the length of the Watson-Crick pairing region between the DNA polynucleotide sequence extending from the nanoscale nucleic acid scaffold (e.g., a DNA origami structure) and the DNA oligonucleotide sequence attached to the plasma nanoparticles is, for example, about 8-30 nucleotides, or about 10-25 nucleotides, or about 12-20 nucleotides.

[0128] As used in this article, the terms “hybridise,” “hybridising,” or similar terms refer to the process by which two complementary or substantially complementary nucleic acid sequences anneal each other according to the Watson-Crick base pairing rule.

[0129] As used herein, the term "substantially complementary" means that two sequences are fully or partially complementary to each other, such that they can hybridize under appropriate stringent hybridization conditions. In other words, the term "substantially complementary" is used to indicate a degree of complementarity or precise pairing sufficient to enable stable and specific binding between two nucleic acid sequences (e.g., between a DNA sequence attached to a plasma nanoparticle and a complementary or substantially complementary DNA sequence within a nanoscale DNA scaffold). It should be understood that a nucleic acid sequence does not need to be 100% complementary to its target or complementary sequence (although this may be a preferred example). For example, a nucleic acid sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% complementary to its target or complementary sequence.

[0130] As described herein, this disclosure is partly based on the ability to immobilize nucleic acid polymerases within unoccupied regions (or “vacant spaces”) of plasma hotspots formed by the plasma nanoantennas described herein, while maintaining the bioactivity of the nucleic acid polymerases, for example, by means of nucleic acid adapters to immobilize the nucleic acid polymerases on the surface of nanoscale nucleic acid scaffolds. In one example, the nucleic acid adapter for tethering the nucleic acid polymerase to the surface of a nanoscale nucleic acid scaffold (e.g., the nanoscale DNA scaffold described herein) is a double-stranded DNA adapter comprising: (i) a DNA polynucleotide covalently or nonvalently bound to the nucleic acid polymerase via amino acids within the nucleic acid polymerase, and (ii) a DNA polynucleotide forming part of or extending from the surface of the nanoscale nucleic acid scaffold, wherein the polynucleotides at (i) and (ii) each contain single-stranded DNA sequences that are complementary or substantially complementary to each other and capable of hybridizing with each other. The polynucleotide bound to the nucleic acid polymerase may contain a peptide tag that is in turn bound to the nucleic acid polymerase via heteropeptide bonds formed between amino acids within the nucleic acid polymerase and amino acids within the peptide tag. In this example, the inventors employed Haterm et al. (2019). Int. J. Mol. Sci The SpyCatcher-SpyTag bioconjugation system described in 20(9):2129 involves attaching a DNA polynucleotide of a double-stranded DNA linker to a nucleic acid polymerase. According to examples using the SpyCatcher-SpyTag system, the polymerase is modified to express the SpyCatcher protein on its surface. The homologous SpyTag is then conjugated to the polynucleotide using any method known in the art. Methods for preparing oligonucleotide-peptide conjugates are known in the art, such as those described in Klabenkova et al. (2021). MoleculesThe methods described in 26(17):5420 include (but are not limited to) conjugation via thioether or disulfide bonds, native linkage, oxime linkage, thiazolyl or hydrazone linkage, amine bond formation, click chemistry, Diels-Alder reaction, and thiol-maleimide conjugation. However, in one specific instance, the DNA polynucleotide is modified to contain maleimide, and SpyTag is conjugated to the DNA polynucleotide via thiol-maleimide conjugation (e.g., as described in the examples herein). Once the DNA polynucleotide containing SpyTag is generated, it is contacted with a polymerase that has been modified to express the SpyCatcher protein. Upon recognition, SpyCatcher and SpyTag form a covalent isopeptide bond between the lysine side chain in SpyCatcher and the aspartic side chain in SpyTag, thereby binding the polymerase to the DNA polynucleotide of the double-stranded DNA linker. Although the SpyCatcher-SpyTag system has been used herein, those skilled in the art will understand that alternative enzyme-mediated conjugation systems (including variant Catcher-Tag systems) can also be employed, and that the covalent binding of polymerase and DNA polynucleotides of double-stranded DNA linkers can rely on alternative amino acid combinations known for forming isopeptide bonds. Those skilled in the art will also be aware of alternative chemical methods that can be used for protein ligation and bioconjugation, which are also considered herein.

[0131] In some instances, the amino acid that binds to the polynucleotide linker in the polymerase is located in the N-terminal domain of the polymerase.

[0132] Each DNA polynucleotide contained in a double-stranded DNA adapter, which is bound to or immobilized by a nucleic acid polymerase on a nanoscale nucleic acid scaffold, contains a region of sufficient length and complementarity to hybridize with each other. The length of each DNA polynucleotide contained in the double-stranded DNA adapter can be from about 20 nucleotides to about 50 nucleotides (e.g., from about 20 to about 30 nucleotides, or from about 20 to about 40 nucleotides, or from about 30 to about 40 nucleotides). In one example, the length of the DNA polynucleotide in the double-stranded DNA adapter is from about 20 to about 30 nucleotides. Preferably, the length of the Watson-Crick pairing region between the corresponding DNA polynucleotides in the double-stranded DNA adapter, which is bound to or immobilized by a polymerase on a nanoscale nucleic acid scaffold, is from about 8 to 30 nucleotides, or from about 10 to 25 nucleotides, or from about 12 to 20 nucleotides. In this respect, those skilled in the art can adjust the length of the Watson-Crick pairing region in the double-stranded DNA adapter to achieve the desired binding specificity and / or binding strength and / or desired hybridization efficiency.

[0133] The DNA polynucleotides contained in double-stranded DNA linkers that enable nucleic acid polymerase binding or immobilization on nanoscale nucleic acid scaffolds may contain one or more modifications. Suitable modifications are known in the art. These include, for example, azo modifications known in the art, which can be used to achieve photoresponsiveness and reversible binding and dissociation of the corresponding DNA sequence. Other modifications include modifications that modulate the strength of interactions between the corresponding DNA sequences, such as scaffold modifications that alter surface charge (e.g., peptide nucleic acids (PNAs)) or structural flexibility to induce hybridization efficiency (e.g., locked nucleic acids (LNAs)). Further modifications include the use of photolytically cleavable structures known in the art.

[0134] In each of the above-described examples of the plasma nanoantennas of this disclosure, the nucleic acid polymerase may be a DNA polymerase.

[0135] As used herein, the term "DNA polymerase" or "DNA polymerase molecule" refers to an enzyme that synthesizes a new DNA strand in the 5' to 3' direction, starting with a primer that hybridizes to a DNA template strand. DNA polymerases are well known and commercially available in the art. DNA polymerase "reads" the template in the 3'→5' direction and adds single nucleotides (bases) to the new or nascent strand in the 5'→3' direction. The polymerase requires a 3'-OH group on the primer to initiate the extension of the new DNA strand. During the synthesis of a new DNA strand by DNA polymerase, single nucleotides (dNTPs, or dATP, dCTP, dTTP, dGTP, or A, C, T, G) are iteratively added to the extending DNA molecule. The specific base (A, C, T, or G) depends on the sequence of the template DNA, such that the new base hybridizes to the nucleotides on the template strand via the Watson-Crick interaction. DNA polymerase cycles between "open" and "closed" conformations. When bound to a primer-template DNA complex, DNA polymerase is in the open conformation. Once a new nucleotide enters the active site, the polymerase will rotate to the closed conformation.

[0136] 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, but most can be classified into families A, B, and C. Family A polymerases are mostly single-stranded proteins that can possess multiple enzymatic functions, including polymerase activity, 3' to 5' exonuclease activity, and 5' to 3' exonuclease activity. Family B polymerases typically have a catalytic domain, possessing polymerase activity and 3' to 5' exonuclease activity, as well as a cofactor. Family C polymerases are typically multi-subunit proteins, possessing polymerization activity and 3' to 5' exonuclease activity. In *Escherichia coli* (… E. coliIn PCR, three types of DNA polymerases have been identified: DNA polymerase I, II, and III (similar to families A, B, and C, respectively). In eukaryotic cells, three distinct family B DNA polymerases (DNA polymerase α, δ, and ∈) are involved in nuclear replication, while family A polymerases (polymerase γ) are used for mitochondrial DNA replication. Other types of DNA polymerases include bacteriophage polymerases. Any one of these polymerases, all or part of a combination of these polymerases, and chimeras or hybrids of two or more of these polymerases or their equivalents can be used to form part or all of the DNA polymerase molecule for the plasma nanoantenna described herein.

[0137] Examples of available DNA polymerases include, but are not limited to: phi-29, Taq, T7, Escherichia coli Klenow (from DNA polymerase I), Escherichia coli DNA polymerase III, and Bacillus stearothermophilus (…). Baccilus stearothermophilus DNA polymerases (Bst) can also be genetically engineered, for example, into hybrids (e.g., Phusion DNA polymerase, in which a domain with a strong affinity for double-stranded DNA is fused to the DNA polymerase to enhance its continuous synthesis capacity). Many useful DNA polymerases are commercially available (e.g., T7 DNA polymerase, Sequenase 2.0™). Polymerases with high continuous synthesis capacity include phi29 and T7 DNA polymerases, as well as Moloney murine leukemia virus (M-MLV) reverse transcriptase. Those skilled in the art will understand that DNA polymerases are structurally similar and that recombinant hybrid polymerases can be engineered using homologous domains from different polymerases.

[0138] In some instances, to avoid potential variations in the incorporation efficiency of fluorescently labeled dNTPs, DNA polymerases and labels that maximize incorporation efficiency and minimize incorporation variability can be used. Some DNA polymerases are capable of efficiently incorporating fluorescently labeled dNTPs along the DNA template, resulting in highly uniform fragments unaffected by sequence background, and these polymerases are used in most automated Sanger / dideoxy-based methods utilizing capillary array DNA sequencers. Many natural and engineered DNA polymerases can incorporate fluorescently labeled 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 et al., Anal Biochem 320, 55-65 (2003); Anderson et al., Biotechniques 38, 257-264 (2005).

[0139] As described above, DNA polymerases can be modified to express amino acid sequences (e.g., CatcherTag or other binding motifs) that can bind to homopeptide tags (e.g., SpyTag or other binding motif-recognized tags) of DNA polynucleotides conjugated to nucleic acid adapters (e.g., double-stranded DNA adapters) described herein, for anchoring the polymerase to the surface of a nanoscale nucleic acid scaffold.

[0140] In some instances, the plasma nanoantennas described herein can be mounted on a substrate via their nucleic acid scaffolds. In specific instances, the substrate is a solid substrate. For example, the plasma nanoantennas described herein can be mounted on glass or silica. For example, the plasma nanoantennas described herein can be mounted on a glass or silica slide or a glass or silica chip. However, other solid substrates are known to those skilled in the art, and these substrates are also considered herein. In some instances, the substrate may be coated with an antifouling material.

[0141] Plasma nanoantenna array This disclosure also provides an array comprising a plurality of plasma nanoantennas described herein. According to this example, the plurality of plasma nanoantennas of this disclosure can be fixed to a substrate. For example, each plasma nanoantenna in the array can be fixed to a solid substrate by its nucleic acid scaffold. Each plasma nanoantenna can be fixed to a solid substrate by a nanoscale DNA scaffold using methods known in the art. For example, the nanoscale DNA scaffold can be fixed to a solid substrate by a DNA adapter or a DNA origami structure. However, those skilled in the art will understand that there are many methods for fixing bioanalytes (such as the plasma nanoantennas of this disclosure) to a substrate, whether covalently or non-covalently via adapter portions or tethered to immobilized portions. These methods are well known in the fields of solid-phase synthesis and microarrays (Beier et al., Nucleic Acids Res. 27: 1970-1-977 (1999)). Non-limiting exemplary binding portions for attaching nucleic acids and proteins to solid supports include streptavidin or avidin / biotin linkages, carbamate linkages, ester linkages, amides, thioesters, (N)-functionalized thiourea, functionalized maleimides, amino groups, disulfide bonds, amides, hydrazone linkages, etc. Furthermore, silyl groups can be directly attached to nucleic acids attached to a substrate (such as glass) using methods known in the art. Other immobilization methods are provided in U.S. Patent Application Serials 11 / 645,125 and 11 / 645,135 (both filed December 21, 2006) and U.S. Patent Publication No. 20080199932, all of which are incorporated herein by reference in their entirety for all purposes. In a specific, non-limiting example, each plasma nanoantenna is fixed to the surface of a substrate (e.g., a glass or silica slide) using an avidin / biotin connector, wherein biotin molecules are coupled to each plasma nanoantenna (e.g., optionally via a connector), and the nanoantennas are fixed to the substrate by surface-fixed avidin (e.g., streptavidin).

[0142] The array may contain multiple plasmonic nanoantennas distributed on the substrate at a desired density and pattern spacing (e.g., at regular intervals).

[0143] The substrate can be formed of any suitable material to which the plasmonic nanoantenna can be covalently or non-covalently attached. Materials include, for example, glass, nylon, carbohydrates (such as dextran), plastics (such as polystyrene or polypropylene), polyacrylamide, latex, silicon, paper, metals (such as gold, chromium, titanium, or tin), titanium oxide, tin oxide, or cellulose. According to one specific example, the substrate is glass, such as a glass slide or chip. Any suitable glass can be used, but the substrate preferably has at least one silica surface layer. In another specific example, the substrate is a flexible plastic. For example, the flexible plastic may contain nitrocellulose acetate. The flexible plastic substrate may also contain flexible plastic strips. Using a flexible plastic substrate improves portability and makes the array easier to connect to other devices, such as being wound around optical fibers and cables to improve sensitivity.

[0144] The substrate can also be provided in any desired form, including chips, beads, holes, flow cells, nanopores, capillaries, glass slides, wafers, filters, fibers, porous media, porous nanotubes, or columns.

[0145] In some instances, assays can be configured to simultaneously measure (e.g., sequence) two or more samples. For this purpose, a solid substrate immobilized with plasmonic nanoantennas can be divided into multiple regions, each containing a unique array of plasmonic nanoantennas. For example, the substrate can be a microplate or microchannel plate. This microplate or microchannel plate can also be configured to be read by a standard microplate reader (e.g., a fluorometer).

[0146] In some instances, the substrate surface can be modified to allow or enhance the covalent or non-covalent attachment of the plasmonic nanoantennas described herein. The substrate and method used for attaching the plasmonic nanoantennas thereto are preferably stable and capable of withstanding repeated bonding, washing, imaging, and elution steps, if desired. In some instances, the substrate surface can be modified to carry a positive or negative charge. In some instances, the substrate surface can be functionalized by modification with specific functional groups (such as maleic acid or succinic acid moieties) or derivatized by modification with chemically reactive groups (such as amino, mercapto, or acrylate groups), such as through silanization. Suitable silane reagents include aminopropyltrimethoxysilane, aminopropyltriethoxysilane, and 4-aminobutyltriethoxysilane. The surface can be functionalized with N-hydroxysuccinimide (NHS) functional groups. The glass surface can also be derivatized with other reactive groups (such as acrylates or epoxy resins), using, for example, epoxysilanes, acrylate silanes, or acrylamide silanes.

[0147] In some instances, the solid substrate can be modified to reduce nonspecific adhesion of the plasmonic nanoantenna to the solid substrate surface. In some instances, the solid support can be modified to reduce nonspecific binding of biological and / or chemical entities to the substrate surface. For example, the substrate surface can be treated with an antifouling agent to prevent nonspecific adsorption to the substrate surface. Suitable antifouling agents are known in the art, and these agents are also considered herein. However, in one specific instance, the substrate surface is treated with APTES. In some instances, the substrate and / or the substrate surface can be passivated. For example, the passivation layer can comprise diamond-like carbon, hexamethyldisilazane, polytetrafluoroethylene, fluorocarbons, polymers (such as polyethylene glycol (PEG)), and / or parylene. In some instances, the substrate can be passivated by attaching polyethylene glycol (PEG) molecules across the substrate. In some instances, the passivation component can be non-covalently bound to the substrate surface. For example, the substrate can be passivated by coating with bovine serum albumin (BSA) or casein (e.g., from dried milk powder).

[0148] In some instances, the solid substrate can be modified across the entire surface of the plasmonic nanoantenna to be attached. In other instances, the solid substrate surface can include modified regions to allow for the attachment of the plasmonic nanoantenna and unmodified regions. Alternatively or additionally, the solid substrate surface can include modified regions to reduce the attachment of the plasmonic nanoantenna and unmodified regions. Alternatively or additionally, the solid substrate surface can include modified regions to increase the attachment of the plasmonic nanoantenna and modified regions to reduce the attachment of the plasmonic nanoantenna. In some cases, the attachment sites of the plasmonic nanoantenna can be generated in an array, such as an ordered array.

[0149] Ordered arrays of attachment sites can be fabricated, for example, by methods such as photolithography, dip-pen nanolithography, nanoimprint lithography, nanosphere lithography, cluster lithography, nanopillar arrays, nanowire lithography, scanning probe lithography, thermochemical lithography, thermal scanning probe lithography, localized oxidation nanolithography, molecular self-assembly, template lithography, or electron beam lithography. The attachment sites in the ordered array can be positioned at arbitrary density and / or configuration. For example, attachment sites in an ordered array can be positioned such that each attachment site is less than 20 nm, or about 20 nm, or about 50 nm, or about 75 nm, or about 100 nm, or about 125 nm, or about 150 nm, or about 175 nm, or about 200 nm, or about 225 nm, or about 250 nm, or about 275 nm, or about 300 nm, or about 325 nm, or about 350 nm, or about 375 nm, or about 400 nm, or about 425 nm, or about 450 nm, or about 475 nm, or about 500 nm, or about 525 nm, or about 550 nm, or about 575 nm, or about 600 nm, or about 625 nm, or about 650 nm, or about 675 nm, or about 700 nm, or about 725 nm, or about 750 nm, or about 775 nm, or about 800 nm. nm, or about 825 nm, or about 850 nm, or about 875 nm, or about 900 nm, or about 925 nm, or about 950 nm, or about 975 nm, or about 1000 nm, or about 1025 nm, or about 1050 nm, or about 1075 nm, or about 1100 nm, or about 1125 nm, or about 1150 nm, or about 1175 nm, or about 1200 nm, or about 1225 nm, or about 1250 nm, or about 1275 nm, or about 1300 nm, or about 1325 nm, or about 1350 nm, or about 1375 nm, or about 1400 nm, or about 1425 nm, or about 1450 nm, or about 1475 nm, or about 1500 nm, or about 1525 nm, or about 1550 nm, or about 1575 nm nm, or about 1600 nm, or about 1625 nm, or about 1650 nm, or about 1675 nm, or about 1700 nm, or about 1725 nm, or about 1750 nm, or about 1775 nm, or about 1800 nm, or about 1825 nm, or about 1850 nm, or about 1875 nm, or about 1900 nm, or about 1925 nm, or about 1950 nm, or about 1975 nm, or about 2000 nm, or more than 2000 nm.

[0150] In some instances, the spacing of attachment sites on the solid substrate surface can be selected based on the size of the plasma nanoantenna to be attached.

[0151] In some instances, less than 30% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas. For example, less than about 25% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas. For example, less than about 20% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas. For example, less than about 15% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas. For example, less than about 10% of the plasmonic nanoantennas in the array are clustered into aggregates of two or more plasmonic nanoantennas.

[0152] In some instances, the substrate may be opaque. In some instances, the substrate may be transparent at one or more wavelengths. In some instances, the substrate may be partially transparent, or transparent in certain regions. For example, a solid substrate may be opaque in unfunctionalized regions and transparent in functionalized regions.

[0153] DNA sequencing methods The inventors have demonstrated for the first time that DNA polymerase molecules can be located in regions of enhanced electric fields when sequencing individual DNA molecules, thereby enabling the detection of the incorporation of specific fluorescently labeled dNTPs into the elongating DNA strand by the DNA polymerase during DNA synthesis.

[0154] Therefore, in one instance, this disclosure provides a method for sequencing nucleic acid analytes, the method comprising: (I) Under certain time and conditions, a nucleic acid polymerase is contacted with a nucleic acid analyte and a labeled nucleotide, such that the labeled nucleotide is sequentially incorporated by the polymerase into a polynucleotide having a sequence complementary to the polynucleotide sequence of the nucleic acid analyte, wherein the polymerase is located within an electric field-enhanced region; and each labeled nucleotide comprises: (i) Adenine nucleotide (A), guanine nucleotide (G), thymine nucleotide (T) or cytosine nucleotide (C). (ii) Fluorescein, and (iii) Polyphosphate linkers that bind nucleotides to fluorophores; In this group, A, G, T, and C are each independently linked to the fluorophore via polyphosphate. When the fluorophore linked to the corresponding nucleotide is excited, each of the labeled nucleotides A, G, T, and C exhibits a unique fluorescence emission characteristic, and The unique fluorescence emission characteristics of the labeled nucleotides are enhanced when incorporated by nucleic acid polymerase into a sequence complementary to the polynucleotide sequence of the nucleic acid analyte; and (II) The sequence of the nucleic acid analyte is determined by detecting the sequence of enhanced unique fluorescence emission features when the labeled nucleotides are sequentially incorporated by a nucleic acid polymerase into a polynucleotide having a sequence complementary to the polynucleotide sequence of the nucleic acid analyte.

[0155] To enable the fluorophore attached to the nucleotide to emit a fluorescent signal upon co-localization with the polymerase, an electromagnetic energy source must be provided to the sequencing reaction. Therefore, this method includes providing an electromagnetic energy source to the labeled nucleotide as it co-localizes with the DNA polymerase. In one example, the electromagnetic energy is provided by one or more lasers. The one or more lasers are configured to provide electromagnetic energy (e.g., light) to the labeled nucleotide at a wavelength corresponding to the excitation wavelength of the fluorophore attached to the nucleotide. As used herein, the phrase "field-enhanced region" refers to a region or extent where the electric field is enhanced relative to the electric field outside the defined region. In some cases, and in the case of DNA sequencing, the enhancement of the electric field is due to plasmon resonance induced by the incident oscillating electric field (e.g., light).

[0156] In some instances, the electric field enhancement region is generated by a plasma hotspot, which is generated by the plasma nanoantenna described herein. Therefore, in some instances, the method includes using the plasma nanoantenna described herein to sequence a nucleic acid analyte according to the method described herein. According to this example, step (I) of the method includes contacting the nucleic acid analyte and a labeled nucleotide with the plasma nanoantenna described herein under certain time and conditions, the contact time and conditions being sufficient to allow the labeled nucleotide to be sequentially incorporated by a nucleic acid polymerase, wherein the polymerase binds to a nanoscale nucleic acid (e.g., DNA) scaffold and is located in a region of the plasma hotspot not occupied by the nanoscale nucleic acid scaffold.

[0157] In one example, the method includes contacting a nucleic acid polymerase with a nucleic acid analyte in the presence of each of the labeled nucleotides A, G, T, and C. In this regard, the method preferably includes detecting the incorporation of each type of naturally occurring nucleotide and allows differentiation of the incorporation of each nucleotide from the incorporation of other types of nucleotides, thereby providing the nucleotide sequence of the nucleic acid analyte. In other examples, the nucleotides of the labeled nucleotides may be further selected from synthetic nucleotides. In one example, the synthetic nucleotides are selected from 5-methylcytidine, N-methylcytidine, and N6-methyladenosine. Therefore, in determining the nucleotide sequence of the nucleic acid analyte, this method can allow differentiation of the incorporation of the synthetic nucleotides described herein from the incorporation of other types of nucleotides.

[0158] The method also includes contacting the nucleic acid analyte (i.e., DNA molecule) with an oligonucleotide primer that is capable of hybridizing specifically with a region of DNA, thereby initiating synthesis via a nucleic acid polymerase (e.g., DNA polymerase).

[0159] In the context of DNA sequencing methods, the terms "primer," "oligonucleotide primer," or similar terms should be understood to mean an oligonucleotide or nucleic acid fragment capable of specifically hybridizing with a template DNA sequence of a DNA molecule, thereby enabling DNA polymerase to initiate the synthesis of such fragments. Primers can be of any length, depending on the specific technique used. However, primers used for DNA synthesis in DNA polymerase reactions (e.g., PCR) are typically between 10 and 40 nucleotides in length. Those skilled in the art can readily design primers with suitable complementarity that specifically hybridize with the target template DNA sequence using methods and software known in the art. In some instances, oligonucleotide primers are designed to be substantially complementary to a universal adaptor template sequence that has been attached (e.g., by ligation) to the 3' end of the template DNA sequence. In other instances, oligonucleotide primers are designed to be substantially complementary to hairpin adaptor sequences that have been attached (e.g., by ligation) to both the 3' and 5' ends of the template DNA sequence. Using universal primers means that only one primer sequence is needed to initiate the synthesis of any number of DNA molecules. Based on examples of preparing libraries of DNA molecules or fragments for sequencing, each of those DNA molecules or fragments may have a universal adaptor template sequence attached to (e.g., by linking) the 3' end, thereby providing a template for specific hybridization to universal primers.

[0160] In the context of hybridization, the terms "selectively" or "specifically" refer to the binding, double-stranding, or hybridization of an oligonucleotide primer to a specific (and often predetermined) location in the template DNA sequence with an affinity (e.g., under more stringent conditions) higher than that with binding, double-stranding, or hybridization to other (non-specific) locations in the template DNA sequence. Those skilled in the art will understand that specific hybridization between nucleotides typically relies on Watson-Crick pairing between complementary nucleotide sequences.

[0161] Nucleic acid polymerases are preferably DNA polymerases. As used herein, the term "DNA polymerase" refers to an enzyme that synthesizes a new DNA strand in the 5' to 3' direction, starting with a primer that hybridizes to a DNA template strand. DNA polymerase "reads" the template in the 3'→5' direction and adds single nucleotides (bases) to the new or nascent strand in the 5'→3' direction. The polymerase requires a 3'-OH group on the primer to initiate the extension of the new DNA strand. During the synthesis of a new DNA strand by DNA polymerase, single nucleotides (dNTPs, or dATP, dCTP, dTTP, dGTP, or A, C, T, G) are iteratively added to the extended DNA molecule. The specific base (A, C, T, or G) depends on the sequence of the template DNA, allowing the new base to hybridize with nucleotides on the template strand via the Watson-Crick interaction. DNA polymerase cycles between "open" and "closed" conformations. When bound to the primer-template DNA complex, DNA polymerase is in the open conformation. Once a new nucleotide enters the active site, the polymerase cycles to the closed conformation. DNA polymerases are well-known enzymes in the art, and many are commercially available. The exemplary DNA polymerases that can be used in the methods of this disclosure have been described above in the context of plasma nanoantennas and should be considered, unless otherwise stated, to be applicable to each instance of the described sequencing method with necessary modifications to the details.

[0162] As used herein, the term "labeled nucleotide" refers to any nucleotide (including naturally occurring and non-naturally occurring nucleotides or "synthetic nucleotides") directly or indirectly attached to a fluorophore. The terms "fluorophore," "fluorescent label," or similar terms refer to the signal transduction portion of a molecule that conveys information through the fluorescent absorption and / or emission properties of one or more molecules. Such fluorescence properties include fluorescence intensity, fluorescence lifetime, emission spectral characteristics, energy transfer, etc.

[0163] The fluorophore binds to the nucleotide via a polyphosphate linker. In one instance, the polyphosphate is a triphosphate, tetraphosphate, pentaphosphate, or hexaphosphate. For example, the polyphosphate is a triphosphate. For example, the polyphosphate is a tetraphosphate. For example, the polyphosphate is a pentaphosphate. For example, the polyphosphate is a hexaphosphate.

[0164] In some instances, the fluorophore binds to the phosphate group furthest from the nucleotide.

[0165] Exemplary fluorophores that can be conjugated to or attached to nucleotides used in sequencing methods include, but are not limited to, fluorescein / Oregon Green, FITC, 6-carboxyfluorescein, tetramethylrhodamine, Texas Red, dansyl, Alexa Fluor 488, BODIPY FL, fluorescein yellow, and Alexa Fluor 405 / Cascade Blue fluorophores.

[0166] Commercially available fluorescent nucleotide analogs that can be easily incorporated into DNA molecules during DNA polymerase synthesis include, for example, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY™ FL-14-dUTP, BODIPY™ TMR-14-dUTP, BODIPY™ TR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™ 630 / 650-14-dUTP, and BODIPY™ 650 / 665-14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXA FLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP, ALEXA FLUOR™ 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADE BLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP, RHODAMINEGREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP,ALEXA FLUOR™ 546-14-UTP (MolecularProbes, Inc., Oreg.). There are protocols for custom synthesis of nucleotides with additional fluorophores. Henegariu et al., “Custom Fluorescent-Nucleotide Synthesis as an Alternative Method for Nucleic Acid Labeling,” Nature Biotechnol 18:345-348 (2000).

[0167] Other fluorophores that can be used for post-synthetic attachment to dNTPs include: ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, dansyl, and lissamine (Rhodamine B). Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, DYLIGHT™ DYES (e.g., DYLIGHT™ 405, DYLIGHT™ 488, DYLIGHT™ 549, DYLIGHT™ 594, DYLIGHT™ 633, DYLIGHT™ 649, DYLIGHT™ 680, DYLIGHT™ 750, DYLIGHT™ 800, etc.) (available from Thermo Fisher Scientific, Rockford, Ill.), Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), and Cy2, Cy3.5, Cy5.5, and Cy7 (available from Amersham Biosciences, Piscataway, NJ USA, etc.).

[0168] Nucleotide-linked fluorophores can have lower quantum efficiency and / or yield. In this respect, weakly emitting fluorophores with low quantum yield may benefit from strong fluorescence enhancement within the plasmon resonance hotspot compared to strongly emitting fluorophores. Using fluorophores with low quantum yield helps reduce background noise from fluorescently labeled nucleotides outside the electric field enhancement region that are not incorporated into the new DNA strand.

[0169] As used herein, the term "fluorescence emission characteristic" refers to the cumulative fluorescence properties of a labeled nucleotide when excited during the sequencing methods described herein, including but not limited to peak emission wavelength, fluorescence emission intensity, and fluorescence emission duration. Therefore, a "unique fluorescence emission characteristic" is a feature that distinguishes a labeled nucleotide from another fluorescence emission characteristic when excited. For example, a "unique fluorescence emission characteristic" as described herein could be the fluorescence emission characteristic of a labeled nucleotide that can distinguish it from another labeled nucleotide used in the sequencing methods described herein based on one or more of the following differences: difference in peak emission wavelength, difference in fluorescence emission intensity, difference in fluorescence emission duration, difference in duration between consecutive fluorescence emissions, or any combination thereof.

[0170] In some instances, unique fluorescence emission characteristics are based on peak emission wavelengths, and two or more types (e.g., 2, 3, or 4) of labeled nucleotides can be distinguished based on differences in their peak emission wavelengths. In one instance, each different type of nucleotide is associated with a fluorophore having a unique peak emission wavelength. In any instance where labeled nucleotides can be distinguished based on differences in their peak emission wavelengths, the peak emission wavelengths of the labeled nucleotides are 10 nm or more apart from each other. For example, the peak emission wavelengths may be about 25 nm or more apart from each other. For example, the peak emission wavelengths may be about 50 nm or more apart from each other. For example, the peak emission wavelengths may be about 75 nm or more apart from each other. For example, the peak emission wavelengths may be about 100 nm or more apart from each other. In some instances, each different type of nucleotide is associated with a fluorophore having a unique peak emission wavelength, and the peak emission wavelengths of the fluorophores are about 100 nm or more apart from each other.

[0171] Each labeled nucleotide may include a fluorophore with a peak emission wavelength independently selected from emission wavelengths in the visible spectrum, ultraviolet (UV) spectrum, infrared (IR) spectrum, and near-infrared spectrum. In one example, one or more fluorophores, or the peak emission wavelength of each fluorophore, is independently selected from emission wavelengths in the range of about 350 nm to 850 nm.

[0172] Alternatively or additionally, different types of nucleotides can be distinguished from one another based on differences in fluorescence emission intensity. For example, differences in fluorescence intensity can be achieved by varying the amount of fluorophores (e.g., the same fluorophore or different fluorophores) attached to different types of nucleotides. According to this example, two or more types of labeled nucleotides used in this method can be labeled with the same fluorophore and can be distinguished based on differences in fluorescence emission intensity. In one example, two or more types of labeled nucleotides used in this method can be distinguished based on differences in fluorescence emission intensity.

[0173] In another example, different types of nucleotides are distinguished from each other based on differences in fluorescence emission duration. In this regard, it is known that different nucleotides have different residence times when incorporated into an elongating DNA strand during synthesis by DNA polymerase. These differences in residence times affect the fluorescence emission duration of the fluorophore linked to the nucleotide. Therefore, in some instances, the unique fluorescence emission characteristic for identifying a nucleotide during the methods of this disclosure may include the fluorescence emission duration specific to that type of nucleotide, or the duration unique to that type of nucleotide combined with a particular fluorophore. According to this example, two or more types of labeled nucleotides used in the method may be distinguished based on differences in fluorescence emission duration.

[0174] As described herein, the unique fluorescence emission characteristics of the corresponding labeled nucleotide incorporated into the elongating DNA sequence by a nucleic acid polymerase are enhanced relative to the fluorescence emission characteristics of the corresponding labeled nucleotide outside the electric field enhancement region. In one instance, the unique fluorescence emission characteristics of the labeled nucleotide incorporated by the polymerase are enhanced by two times or more (e.g., about three times or more, or about four times or more, or about five times or more, or about six times or more, or about seven times or more, or about eight times or more, or about nine times or more). In some instances, the unique fluorescence emission characteristics of the labeled nucleotide incorporated by polymerase are enhanced by an order of magnitude or more (e.g., about 10 times or more, or about 20 times or more, or about 30 times or more, or about 40 times or more, or about 50 times or more, or about 60 times or more, or about 70 times or more, or about 80 times or more, or about 90 times or more, or about 100 times or more) compared to the fluorescence emission characteristics of the corresponding labeled nucleotide outside the electric field enhancement region. In other instances, the unique fluorescence emission characteristics of the labeled nucleotide incorporated by polymerase are enhanced by at least about 100 times or more (e.g., at least about 150 times, or at least about 200 times, or at least about 250 times, or at least about 300 times, or at least about 350 times, or at least about 400 times, or at least about 450 times, or at least about 500 times, or at least about 550 times, or at least about 600 times, or at least about 650 times, or at least about 700 times, or at least about 750 times, or at least about 800 times, or at least about 850 times, or at least about 900 times, or at least about 950 times, or at least about 1000 times) compared to the fluorescence emission characteristics of the corresponding labeled nucleotide outside the electric field enhancement region.

[0175] In some instances, the method further includes contacting the nucleic acid polymerase with the nucleic acid analyte and the labeled nucleotide in the presence of one or more quenchers. As used herein, a “quenching agent” or “quencher” refers to any fluorescent modification that can attenuate or reduce the emission of light from a fluorophore. This attenuation or reduction of light emission is called “quenching.” Therefore, in some instances, one or more quenchers may be provided to help reduce background noise from fluorophores linked to labeled nucleotides located outside the region of electric field enhancement. In some instances, the quencher is provided in a free, unconjugated form. Quenching agents that can be used in the methods disclosed herein include, but are not limited to: black hole quenching dyes (Biosearch Technologies), such as BHQ-0, BHQ-1, BHQ-2, BHQ-3, and BHQ-10; QSY dye fluorescence quenchers (from Molecular Probes / Invitrogen), such as QSY7, QSY9, QSY21, and QSY35; and other quenchers, such as Dabeyl and Dabsyl; Cy5Q and Cy7Q; and dark cyanine dyes (GE Healthcare), which can be used, for example, with donor fluorescent agents (fluorescein) such as Cy3B, Cy3, or Cy5; DY quenchers (Dyomics), such as DYQ-660 and DYQ-661; and ATTO fluorescence quenchers (ATTO-TEC GmbH), such as ATTO 540Q, 580Q, and 612Q.

[0176] The term "nucleic acid analyte" refers to any nucleic acid molecule whose sequence will be determined using the methods of this disclosure. More broadly, "nucleic acid" refers to a polymer having multiple nucleotide monomers. Nucleic acid analytes can be single-stranded or double-stranded and can be DNA (e.g., complementary DNA, genomic DNA, or mitochondrial DNA), RNA, or a hybrid polymer (e.g., DNA / RNA). The term "nucleic acid" does not refer to a polymer of any particular length. Rather, nucleic acids can be of any length composed of nucleotides, for example, 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, or greater than about 1,000,000 bases or more.

[0177] The nucleic acid analytes to be sequenced can be obtained from any target source and can include DNA, mRNA, and their analogues, derivatives, and conjugates. They can be isolated from cells, cell cultures, tissue samples, body fluids, viral samples, genomic 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 obtain the nucleic acids to be sequenced, as these manipulations do not render the nucleic acids unsuitable for the subsequent sequencing described herein. It should be understood that such nucleic acids may contain modified, non-canonical, and / or non-natural nucleotides or nucleotide analogues, many of which are described in U.S. Patent Application Serial No. 12 / 945,767, filed November 12, 2010, the entire contents of which are incorporated herein by reference for all purposes. Therefore, the methods disclosed herein can 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.

[0178] In each of the above examples, the nucleic acid analyte is sequenced by determining the sequence of nucleotides in the DNA molecule. In one example, the DNA is genomic DNA. In another example, the DNA is complementary DNA (cDNA) derived from RNA. Depending on the example where the nucleic acid analyte is cDNA, the method may include determining the RNA sequence based on the sequence of the corresponding cDNA.

[0179] In each of the above examples, the nucleic acid analyte may undergo one or more processing steps before being localized by the plasmonic nanoantenna. These processing steps may include, but are not limited to: purification steps, reverse transcription (e.g., in the case of RNA), fragmentation to obtain template DNA of the desired size, enrichment steps (e.g., targeting a specific sequence or structure), removal steps (e.g., to remove unwanted sequences), amplification steps (e.g., to increase the amount of template DNA), ligation steps (e.g., to attach adaptors and other target sequences), and combinations thereof. The choice of processing steps and techniques used may vary depending on the application. In some examples, combinations of these processing steps may be employed to prepare one or more libraries (e.g., DNA template libraries) for sequencing using the methods of this disclosure. Numerous protocols and methods for preparing nucleic acid samples for sequencing reactions are documented in the literature, and those skilled in the art can readily determine the necessary steps and methods.

[0180] In some instances, the nucleic acid analyte is a double-stranded DNA molecule that can be denatured to produce a single-stranded DNA molecule (i.e., "target DNA sequence," "template DNA," or "template DNA sequence"), which can be used by DNA polymerase to synthesize a new strand. The terms "target DNA" or "template DNA" refer to a DNA segment that can be sequenced according to this method. This term encompasses the two complementary strands of the target DNA molecule. Those skilled in the art should understand that, due to the well-known Watson-Crick base pairing, the sequence of one strand of the target DNA can be used to infer the sequence of the other strand. The target DNA can be attached to one or more oligonucleotide primers or adaptors, for example, to facilitate synthesis. However, in other instances, the double-stranded DNA molecule is not denatured but remains in its double-stranded form.

[0181] Hydrodynamic mechanical shearing can be used to fragment or cut DNA molecules (including cDNA, single-stranded DNA, and double-stranded DNA) into different sizes (0.5 kb to 10 kb) with a relatively narrow size distribution (Thorstenson et al., Genome Res 8, 848-855 (1998); Roe, Methods Mol Biol 255, 171-187 (2004)). Methods for fragmenting and / or cutting DNA are known in the art, including, for example, sonication, nebulization, and enzymatic methods. Specialized equipment for limiting the size of DNA cuts is commercially available (e.g., Bioruptor® from Diagenode). These methods are described in detail 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), the entire contents of which are incorporated herein by reference for all purposes.

[0182] The DNA molecule to be sequenced can be of any length. For example, the length of the DNA molecule to be sequenced can be 10 or more nucleotides, such as about 20 nucleotides, or about 50 nucleotides, or about 100 nucleotides, or about 200 nucleotides, or about 300 nucleotides, or about 400 nucleotides, or about 500 nucleotides, or about 1000 nucleotides, or longer (e.g., 2000, 5000, or 10000 or more nucleotides). In some instances, the length of the DNA molecule to be sequenced is about 10-10000 nucleotides. In some instances, the length of the DNA molecule to be sequenced is about 100-5000 nucleotides. In some instances, the length of the DNA molecule to be sequenced is about 200-2000 nucleotides. In some instances, the length of the DNA molecule to be sequenced is about 400-1000 nucleotides. As described in this article, in some instances, larger DNA molecules can be fragmented or cut into various smaller sizes to facilitate sequencing.

[0183] The DNA molecule to be sequenced contains one or more adaptor sequences that provide binding sites for one or more oligonucleotide primers, enabling hybridization and initiation of synthesis via DNA polymerase. Therefore, the method may include the further step of ligating the adaptor to one or more DNA molecules (e.g., a DNA molecular library) being sequenced using the methods described herein. For example, the method may include ligating a universal adaptor sequence to the 3' end of the DNA molecule to be sequenced. In some instances, the method may include ligating a hairpin adaptor to both ends of a double-stranded DNA molecule to provide a sequencing template containing both the complementary and non-complementary strands of a DNA molecule in a single-stranded circular construct, which can be repeatedly sequenced to provide redundant sequencing information from both strands. For single-molecule sequencing reactions, such as SMRT® sequencing from Pacific Biosciences, where sequence data is generated from a single template molecule, a common sequence for the target region is generated from the single sequencing template using statistical analysis of the redundancy information. Further details regarding redundant sequencing and circular sequencing templates are provided, for example, in U.S. Patent Nos. 7,476,503 and 8,153,375, the entire contents of which are incorporated herein by reference for all purposes.

[0184] In other instances, the method may include attaching a hairpin adaptor to only one end of a double-stranded DNA molecule. Attaching a hairpin adaptor to only one end of a double-stranded DNA molecule provides a sequencing template comprising both the complementary and non-complementary strands of a DNA molecule in a single-stranded linear construct, which can be sequenced to provide sequence reads for each strand. Such templates are particularly advantageous in sequencing technologies that prefer single-stranded linear templates, for example, in sequencing technologies using nanopore sensors, which have been described in detail in the prior art and are currently being developed by several companies, including Oxford Nanopore and Genia. However, in nanopore-based sequencing methods, templates with stem-loop adaptors at both ends can also be considered, for example, using a single-stranded loop generated by separating the complementary strand as a template for rolling circle replication, for example, guiding the nascent strand or released phosphate groups into or through the nanopore.

[0185] In each of the above examples, step (II) of the nucleic acid analyte sequencing method includes detecting the sequence of enhanced, unique fluorescence emission characteristics as a labeled nucleotide is sequentially incorporated by a nucleic acid (e.g., DNA) polymerase into a polynucleotide having a sequence complementary to the polynucleotide sequence of the nucleic acid analyte (i.e., the complementary strand). The enhanced, unique fluorescence emission characteristics can be detected by any method 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, light conversion, wide-field fluorescence, single-photon and / or multiphoton excitation, spectral wavelength identification, evanescent wave illumination, scanning two-photon, scanning wide-field two-photon, Nipkow rotating disk, and / or multifocal multiphoton. In some examples, combinations of these methods are used to detect fluorescence. In one example, confocal laser scanning microscopy is used to detect the enhanced, unique fluorescence emission characteristics as a nucleotide is incorporated into the complementary strand by a DNA polymerase. In one example, total internal reflectance (TIR) ​​is used to detect the unique enhanced fluorescence emission feature when a nucleotide is incorporated into the complementary strand by DNA polymerase. In another example, total internal reflectance fluorescence (TIRF) is used to detect the unique enhanced fluorescence emission feature when a nucleotide is incorporated into the complementary strand by DNA polymerase.

[0186] The unique fluorescence emission characteristics emitted from different types of labeled nucleotides can be resolved using any suitable identification method based on: fluorescence resonance energy transfer measurement; light conversion; fluorescence lifetime measurement; polarization; fluorescence lifetime determination; correlation / inverse correlation analysis; Raman spectroscopy; intensity analysis; ratiometric method; time-resolved method; anisotropy; near-field or far-field microscopy; fluorescence recovery after photobleaching (1-RAP); spectral wavelength identification; fluorescence lifetime measurement and separation; fluorophore identification; background suppression; parallel multicolor imaging, or any combination thereof. See, for example, JR Lakowitz 2006, “Principles of Fluorescence Spectroscopy”, 3rd edition. If different nucleotides are labeled with different energy transfer or reporter portions, the different nucleotides bound to and / or incorporated by polymerase can be distinguished by resolving the emission signals. In a specific example, each labeled nucleotide emits a fluorescence signal at a different wavelength, thus distinguishing the unique fluorescence emission characteristics of nucleotide types based on the emission wavelength (e.g., peak emission wavelength).

[0187] To detect the unique fluorescence emission characteristics of each of the different types of labeled nucleotides, the method can be further employed with a multi-fluorescence imaging system capable of detecting multiple unique fluorescence emission characteristics emitted by different types of labeled nucleotides during the sequencing reaction. Such a system may include a dedicated combination of filters for each excitation line and / or each emission band. In one example, the detection system includes tunable excitation and / or tunable emission fluorescence imaging.

[0188] If the method of this disclosure is performed in an array (e.g., the plasma nanoantenna array described herein), the detection system may include an optical system that directs signals emitted from the ordered array to different locations on an array-based detector to detect multiple optical signals from multiple locations. This optical system typically includes gratings and / or wedge prisms for simultaneously directing and separating signals with different spectral characteristics from different addressable locations in the array to different locations on the array-based detector (e.g., a CCD).

[0189] After detecting the sequence of unique fluorescence emission features in step (II), the sequence of the nucleic acid analyte can be determined by associating the sequence of unique fluorescence emission features with the sequence of nucleotides sequentially incorporated into the synthetic DNA.

[0190] Reagent test kit This disclosure also provides kits and reaction mixtures for performing the single nucleic acid molecular sequencing described herein. The components and configurations of the kit may vary, but generally include a plasmonic nanoantenna attached to a substrate as described herein, or a plasmonic nanoantenna array as described herein, and a reaction mixture. The reaction mixture may contain one or more fluorescently labeled nucleotides (e.g., each of dATP, dCTP, dGTP, and dTTP as described herein), one or more buffers (e.g., Tris), various salts (e.g., KCl, NaCl, (NH4)2SO4, MnCl2, zinc salts, MgCl2), and conventional stabilizers, detergents, DMSO, and DTT. The reaction mixture of this disclosure may also contain additives to improve the specificity and efficiency of the polymerase reaction. The reaction mixture may contain oligonucleotide primers for primer DNA synthesis via DNA polymerase. If a universal primer hybridizes to a universal adaptor sequence, the universal primer may (but is not required to) be provided in the reaction mixture. If a custom primer sequence is used, it is preferable, for example, that the primer is provided separately by the user. It should be understood that the kit of this disclosure may contain any combination of the above components.

[0191] The kit disclosed herein may also include instructions for performing the sequencing methods for a single DNA molecule described herein.

[0192] Any discussion of documents, actions, materials, devices, articles or similar content included in this specification shall not be construed as an admission that any or all of these matters constitute part of the prior art or common general knowledge in the relevant field prior to the priority date of each appended claim. Example

[0193] Example 1. General Materials and Methods DNA Origami Design and Synthesis The inventors used caDNAno to design a DNA staple sequence for folding M13mp18 single-stranded DNA templates into DNA origami nanospanno scaffolds.

[0194] FEM simulations used to estimate fluorescence enhancement All simulations were performed in MATLAB 2020b using the toolbox for simulating metallic nanoparticles using the boundary element method (MNPBEM). 22 The optical properties of silver and gold were obtained from Johnson and Christy. 23The dielectric constant of the background medium was set to 1.77 to match the aqueous buffer used in the experiment. To simulate measurements using total internal reflection fluorescence (TIRF) microscopy, a glass substrate with a dielectric constant of 2.25 was added 5 nm below the nanoparticles along the z-axis, with the incident plane wave propagating at a 65° angle relative to the glass layer normal. The emission characteristics of the dye were modeled using an oscillating dipole at a frequency corresponding to the dye's maximum emission wavelength.

[0195] DNA origami synthesis DNA origami synthesis involves folding a single long template DNA, consisting of a single-stranded bacteriophage vector M13mp18, by hybridizing it with approximately 250 short "staple" DNA strands that cross-link the DNA helix at specific locations to form the desired shape. 21,24 The synthesis requires an annealing process to prevent the formation of undesirable kinetically trapped structures and to maximize the yield of well-formed structures conforming to the design. The optimized synthesis conditions are as follows: 1× origami buffer (10 mM Tris pH 8 and 1 mM EDTA) plus 20 mM magnesium chloride (MgCl2), 15 times excess staples (50 times excess extended staples), annealing for 22 hours, with an optimal temperature range of 48–43°C. Details of the temperature gradient for the annealing reaction are shown in Table 1 below.

[0196] Table 1. Temperature gradient for DNA origami synthesis

[0197] Agarose gel electrophoresis Mix 10 μL of 5 nM sample (DNA origami or M13mp18 single-stranded template) or molecular weight standard with 2 μL of loading dye and load onto an agarose gel prepared by dissolving 1.125–3 g of agarose in 150 mL of buffer (with or without 7.5 μL of RedSafe dye). Perform electrophoresis at 70 V for 3 hours in pre-chilled 1X TAE buffer containing 6 mM MgCl2. If the sample is labeled with Alexa Fluorophore, scan the gel using Alexa Fluorophore 647 / 488; if RedSafe is added to the gel, scan using RedSafe dye. After staining with SYBRgold, scan the gel again using SYBRgold.

[0198] DNA origami purification The inventors purified DNA origami by extracting and purifying samples from agarose electrophoresis gels or by precipitating with polyethylene glycol (PEG).

[0199] Gel extraction and purification A 0.75% agarose gel was prepared by dissolving 1.125 g of agarose in 150 mL of 1xTAE buffer containing 11 mM MgCl2, and RedSafe staining was added for visualization. The sample mixed with the loading dye was loaded onto the gel and electrophoresed on ice at 70 V for 2.5 h. After electrophoresis, the DNA origami strips were cut with a clean blade and transferred to a 500 μL Eppendorf tube, centrifuged at maximum speed for 5 min to disrupt the gel. The gel slurry was then transferred to a Freeze'N'Squeeze column and centrifuged at maximum relative centrifugation force (rcf) at 4 °C for 10 min. Finally, the purified DNA origami was collected in the column's accompanying collection tube.

[0200] Polyethylene glycol (PEG) precipitation purification Unpurified DNA origami samples were pipetted into DNA low-binding tubes and brought to a final volume of 400 μL with pre-PEG buffer (1x origami buffer and 20 mM MgCl2, filtered through a 0.45 μm syringe). 400 μL of 2x PEG precipitation buffer (15% (g / mL) PEG8000, 10 mM Tris pH 8, 1 mM EDTA, 500 mM NaCl) was added, and the sample was centrifuged at 21100 RCF for 25 min at 20 °C. Immediately after centrifugation, the supernatant was removed, and the sample was resuspended in 1x origami buffer containing 6 mM MgCl2 (also filtered through a 0.45 μm syringe). After resuspending in 50 μL of 1x origami buffer containing 6 mM MgCl2, the concentration was quantified using Nanodrop. Concentration in nM was calculated by dividing the concentration in ng / μL by 4.8.

[0201] Determining the size of DNA origami Single-particle averaging (SPA) analysis was performed using RELION to accurately determine the size of NanoSpanno. All images used for SPA were acquired using a Tecnai microscope at a consistent magnification of 19,000–38,000x.

[0202] Preparation of carrier network A carbon / Formvar screen was subjected to glow discharge treatment, and a drop of 2% uranium acetate (or uranium formate) staining solution was placed on a paraffin film. A 5 μL droplet of 1 nM sample (undiluted, obtained from gel purification) was applied to the dark side of the screen and immediately aspirated from the screen using a filter paper at the edge. Then, the screen (dark side) was brought into contact with the staining droplet, and the staining droplet was immediately aspirated from the screen using a filter paper. The screen was then air-dried for several minutes.

[0203] Imaging conditions All images used for SPA were acquired using a Tecnai microscope at a consistent magnification of 19,000–38,000x.

[0204] Two-dimensional particle average analysis Single-particle average (SPA) analysis was performed using RELION to accurately determine the size of NanoSpanno.

[0205] Preparation of a grid for cryo-transmission electron microscopy (cryo-TEM) To prepare samples for cryo-electron microscopy (cryo-EM) observation, an aqueous solution is applied to a glow discharge-treated (hydrophilic) porous carbon film supported by an electron microscope (EM) grid. Excess solution is carefully blotted away from one or both sides using filter paper. The blotted grid is then rapidly immersed in a cryogenic medium pre-cooled to liquid nitrogen temperature. This rapid freezing process embeds biomolecules in a thin, amorphous ice film, allowing for observation under a cryo-EM microscope.

[0206] Imaging using cryo-electron transmission microscopy To avoid ice contamination, the frozen grid was mounted on a cryogenic transfer holder equipped with a liquid nitrogen Dewar flask in the cryogenic workstation. The ice-embedded sample was then loaded onto the cryo-electron microscope in a contamination-free frozen state via the cryogenic transfer holder. Images of the pores were captured using low-dose mode. The focus was adjusted, and the target was then exposed at the desired preset magnification.

[0207] DNA-coated spherical gold nanoparticles Materials for using DNA-coated gold nanoparticles • 100 nm gold nanoparticles (AuNP), purchased from Nanopartz • Thioylated DNA, purchased from IDT (Coralville USA) • Phosphate buffer [Preparation method: Mix 980 μL of MQ water with 10 μL of Tween 20 (10%) and 10 μL of potassium phosphate (a mixture of 4:5 of 1M KH2PO4 and 1M K2HPO4)] •10% Tween-20 • 20 mM TCEP: 5.732 mg / mL (in MQW, adjust pH to 3.0) Preparation of thiolized DNA A 25 μM thiol-DNA solution in 10 mM TCEP was prepared by adding 0.5 μL of monothiol-DNA (25 AC, 500 μM, in Milli-Q water) + 5 μL of TCEP (20 mM, pH=3) + 4.5 μL of Milli-Q water. This solution was incubated at room temperature for 1 hour. Then, 25 μL of Milli-Q water was added to bring the total volume to 35 μL.

[0208] Gold nanoparticles coated with thiolized DNA 1 mL of nanoparticle solution (10 pM, with 10 μL of 5x phosphate buffer) was centrifuged at 4500 RCF for 5 min. The supernatant was discarded, and the nanoparticle precipitate was mixed with 35 μL of TCEP-treated DNA, then diluted and mixed in 10 μL of 5x phosphate buffer (total volume 50 μL). The nanoparticle-oligonucleotide mixture was then frozen at -20°C for 2 h, followed by refrigeration at room temperature. The final conditions consisted of: 0.2 nM gold nanoparticles and 5 μM DNA—a 25,000-fold molar excess.

[0209] Excess DNA was removed from DNA-coated gold nanoparticles by repeated precipitation and supernatant removal. The mixture was centrifuged at 4500 RCF for 5 min, the supernatant was discarded, and the particle precipitate was diluted in 10 μL of 5x phosphate buffer, and then diluted in 1 mL of MQW. The precipitation process was repeated 5 times to completely purify the nanoparticles from the free oligonucleotides.

[0210] Protein expression and purification Material • 1000x ampicillin: 100 mg / ml (1 g / 10 mL) •LB agar •LB Broth •1000x IPTG: 1 M (0.238 g / mL) • Klenow buffer: 10 mM Tris HCl pH 8, 50 mM NaCl, 10 mM MgCl2 and 1 mM DTT • Phi29 buffer: 50 mM Tris HCl pH 7.5, 50 mM NaCl, 10 mM MgCl2 and 4 mM DTT •Taq: 10mM Tris HCl pH 8, 50mM NaCl, 1.5mM MgCl2 • 120 mL 1M imidazole • 500 mL lysis buffer: 20 mM Tris pH 7.5, 300 mM NaCl (420 mL for washing buffer) • 500 ml wash buffer: 20 mM Tris pH 7.5, 300 mM NaCl, 20 mM imidazole • 200 mL elution buffer: 20 mM Tris pH 7.5, 300 mM NaCl, 500 mM imidazole • IL SEC buffer: 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT • 100 mL SEC buffer (after fluorescent labeling): 20 mM Tris pH 7.5, 150 mM NaCl • Dialysis buffer: 20 mM Tris pH 7.5, 150 mM NaCl • 0.1M TCEP pH 7 • Alexa 647 fluorophore, 50 nmol aliquots (resuspended in 10 μL DMSO, stock solution concentration 5 mM) plasmid description Three petDuet-1 plasmids were ordered, each containing one of the following recombinant polymerases: Klenow-SpyC, Phi29-SpyC, and Taq-SpyC (Genscript). Each recombinant polymerase construct was cloned into the petDuet-1 vector for expression in E. coli cells.

[0211] Transform T7 E. coli with expression plasmid Ampicillin-containing LB agar plates were prepared by heating 300 ml of Luria broth (LB) agar to a liquid state and then cooling to room temperature. Before solidification, 300 µL of 100 mg / ml ampicillin was added to the LB agar to produce a final concentration of 100 µg / ml ampicillin. The LB agar was then poured into sterilized petri dishes and allowed to solidify.

[0212] 1 μL of plasmid was transformed into 20 μL of T7 *E. coli* expression cells. The cells were first incubated on ice for 30 min, and then 1 μL of the expression plasmid was added. The mixture was then incubated in a 42°C water bath for 10 s, followed by 5 min on ice. 20 μL of the transformed cells were plated onto Luria broth (LB) agar plates containing 100 µg / ml ampicillin and incubated overnight at 37°C. Single colonies containing the expression plasmid were observed.

[0213] Protein expression A single *E. coli* colony was placed in 100 ml of starting culture containing 100 µg / ml ampicillin and incubated overnight at 37°C. Approximately 25 mL of the starting culture was added to 2 L LB broth containing 100 µg / ml ampicillin. The culture was grown on a shaker at 37°C and 180 rpm until the OD600 value reached 0.5–0.6. The temperature was then lowered to 18°C, and IPTG was added to a final concentration of 1 mM to induce protein expression. The cells were then cultured overnight on a track shaker at 180 rpm to produce protein.

[0214] Cells were then centrifuged at 6000 RCF for 20 min at 4°C (centrifuge VX22N, rotor R9A2, VWR). The cell pellet was stored at -80°C for later use. The cell pellet was resuspended in 80 mL of lysis buffer (+1 Complete EDTA tablet + 1 DNase) and sonicated for 3 × 3 min at 50% duty cycle and power output of 7.3 to lyse the cells. Solids were removed by centrifugation, and proteins expressed in the cytoplasm were extracted. The sample was centrifuged at 3600 RCF for 30 min at 4°C. The pellet was discarded, and the cell lysate supernatant was collected for purification (centrifuge VX22N, rotor R9A2, VWR).

[0215] Protein purification using immobilized metal affinity purification (IMAC) Immobilized metal affinity chromatography (IMAC) was performed using 2 x 5 mL HisTrap Fast Flow Crude columns. The HisTrap columns were prepared as follows: first, the column was equilibrated with 30 mL of washing buffer (3 column volumes), then any contaminants were removed with 30 mL of elution buffer, and the column was equilibrated again with 30 mL of washing buffer before loading cell lysis buffer.

[0216] Approximately 75 ml of cell lysate was filtered through a 0.22 μm membrane and then loaded onto a HisTrap column at a flow rate of 1 ml / min. The HisTrap column was then washed with washing buffer (20 ml) to elute bound proteins. A gradient elution was performed at 2 ml / min in 10 column volumes from 20 mM to 500 mM imidazole, and 2 ml fractions were collected.

[0217] Protein purification by size exclusion chromatography (SEC) The elution fractions containing recombinant polymerase purified from IMAC were combined and concentrated to 5 mL using a 30KD Amicon Ultra ultrafiltration centrifuge unit, followed by further purification with SEC. SEC was performed using a HiLoad 16 / 600 Superdex 200 pg column (GE Healthcare). The column was equilibrated with MQW (approximately 150 mL, 1.2 column volumes, 0.9 mL / min, <0.5 MPa). The SEC column was equilibrated with SEC buffer (approximately 150 mL, 1.2 column volumes, 1 mL / min, <0.5 MPa). Protein was eluted from the SEC column using SEC buffer (1.2 column volumes, 150 mL, injection less than 5 mL). The elution fractions were run on SDS-PAGE as described above to determine protein purity. The fractions containing purified polymerase were combined, and the final concentration was determined using Denovix. The recombinant polymerase was stored at -80°C until needed.

[0218] Covalent attachment of fluorescent dyes to proteins The purified protein was dialyzed to remove DTT. Approximately 1 mL of protein (80 μM) was added to a 6 KD-cut dialysis button. The button was placed in 1 L of dialysis buffer and stirred overnight at 4°C, with the buffer replaced once during the incubation period. After dialysis, the concentration was determined by infrared absorption on a Denovix instrument.

[0219] Add 10 μL of TCEP (20 mM, 10 μL) to 400 μL of 50 μM dialyzed protein and incubate on ice for approximately 30 min. Then label the protein with a 5-fold molar excess of Alexa-647 maleimide dye. Add 400 μL of TCEP-containing protein to 20 μL of DMSO solution containing 100 nmol of Alexa-647 dye (5-fold excess). Incubate the mixture overnight on a rotating wheel in the dark. Then purify the excess dye with SEC and replace the buffer with 20 mM Tris pH 7.5 and 150 mM NaCl.

[0220] SDS-PAGE 4-12% SDS-PAGE gels were purchased from Invitrogen. Samples were run at 180V for 30 min in 1x MES buffer. All SDS-PAGE gels used protein molecular weight (MW) standards were SeeBlue Plus2 pre-stained protein standards. After Blue staining, the gels were imaged under UV light to identify which fractions contained pure protein.

[0221] High-performance liquid chromatography (HPLC) Maleimide DNA, DNA-peptide conjugates, and fluorescently labeled dNTPs were purified by high-performance liquid chromatography (HPLC) using a Shimadzu LC-20AT system equipped with an Atlantis RP-HPLC C18 column (150 mm × 4.6 mm, 5 μm). Fractions were automatically collected, and the fraction containing the purified product was evaporated. The resulting residue was dissolved in water and quantitatively analyzed using a Denovix instrument.

[0222] HPLC methods for maleimide DNA and DNA-peptide conjugates The sample was diluted with 0.1M TEAA + 5% ACN, and 100 μL was injected into the HPLC system. The HPLC buffer consisted of HPLC buffer A (containing 0.1M TEAA + 5% ACN) and HPLC buffer B (composed of 0.1M TEAA + 70% ACN). The HPLC procedure was as follows: injection was performed at t = 0 min, with a buffer flow rate of 1 mL / min. The protocol included: pumping in at 0% buffer concentration for 1 min; then increasing the concentration to 100% buffer concentration from 1 to 40 min; and maintaining this 100% buffer concentration until 43 min. Then, at 43.01 min, the concentration was reduced to 0% buffer concentration and maintained until 48 min.

[0223] HPLC methods for fluorophore-labeled dNTPs The sample was diluted with 0.1 M TEAA, and the injection volume was 100 μL. The buffers used in the HPLC method were HPLC buffer A (composed of 0.1 M TEAA) and HPLC buffer B (composed of 100% ACN). The procedure involved running the HPLC method, with injection at t = 0 min, and the buffer flowing at a specific rate of 1 mL / min. The protocol involved pumping in at a 2% buffer concentration for 1 min; then increasing the concentration to 7% buffer concentration from 1 to 15 min, then increasing it to 30% buffer concentration from 15 to 45 min, and finally increasing it to 60% buffer concentration from 45 to 75 min and maintaining it at 60% until 80 min. After 80.01 min, the concentration was reduced back to 2% buffer concentration and maintained for 5.00 min until the controller stopped operation at 85.01 min.

[0224] Synthesis of DNA-peptide conjugates Synthesis and purification of maleimide-DNA To synthesize maleimide-DNA, 2 mg SMCC was resuspended in 300 μL DMF and 10 μL LIPEA was added. Then, 5 μL of 2 mM aminoated DNA (10 nmol) was added to the solution, which was incubated on an orbital shaker at 600 rpm for 45 minutes at room temperature.

[0225] Next, SMCC-DNA was extracted by ethanol precipitation. The solution was divided into three aliquots, each approximately 105 μL. To each aliquot, 100 μL of MilliQ water, 20 μL of 3M pH 5.2 sodium acetate, and 800 μL of ice-cold 100% EtOH were added, and the aliquots were incubated at -80°C for 45 min. The DNA precipitate was centrifuged at 25000 RCF at 4°C for 45 min to form clumps, and the supernatant was removed. The clumps were dried (in a 37°C oven for approximately 15 min) and stored at -20°C.

[0226] The final purification step was performed using high-performance liquid chromatography (HPLC). The precipitate was resuspended in 100 μL MilliQ water, and undissolved precipitate was removed by centrifugation and filtration. The supernatant was loaded onto the HPLC using method 2.10.1 and eluted with an acetonitrile gradient. Typically, 5'-maleimide-15nt DNA and 3'-maleimide-15nt DNA eluted at approximately 15 minutes, while 5'Cy5-3'-maleimide-15nt DNA eluted at approximately 22 minutes. The fraction containing the purified product was evaporated, and the remaining residue was dissolved in water and quantified using UV-Vis spectrophotometry. The product was then aliquoted into 100 pmol / tubes, allowing resuspending in 10 μL MQW to prepare a 10 μM working stock solution. The sample was then dried using a vacuum centrifuge (speedyvac) and stored at -20°C.

[0227] Maleimide DNA conjugation with peptide conjugates Maleimide-DNA was resuspended in 80 μL MilliQ water and 10 μL 200 mM Tris (pH 7.5, 1.5 M NaCl). Then, 10 μL of a 1 mM cysteine-containing peptide was added, and the mixture was incubated at room temperature for 2 h, followed by purification by HPLC. This was done according to the procedure described in Example 1, titled "..." HPLC methods for maleimide DNA and DNA-peptide conjugates As described in the section, 100 μL of sample was loaded onto HPLC.

[0228] Typically, 5'-SpyTag-15nt DNA and 3'-SpyTag-15nt DNA elute at approximately 17 minutes, while 5'Cy5-3' maleimide-15nt DNA elutes at approximately 20 minutes. The fraction containing the purified product is evaporated, and the remaining residue is dissolved in water and quantified using UV-Vis spectrophotometry. The product is then aliquoted into 100 pmol / tubes and resuspended in 10 μL MQW to prepare a 10 μM working stock solution. The sample is then dried using a vacuum centrifuge and stored at -20°C.

[0229] Synthesis of DNA-protein conjugates The purified SpyTag-DNA can then be combined with the purified polymerase SpyCatcher to generate a polymerase covalently linked to a DNA strand with the desired sequence, enabling specific immobilization within the cavity of the DNA origami nanoantenna.

[0230] The buffer conditions are as follows: • Klenow 10x reaction buffer: 100 mM Tris pH 8, 0.5 M NaCl, 100 mM MgCl2, 10 mM M DTT • Binding and elution buffers: • 1M imidazole solution (68 mg / mL) • Binding and washing buffers: 20 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole • Elution buffer: 20 mM Tris pH 7.5, 300 mM NaCl, 500 mM imidazole SpyTag-DNA was incubated with SpyCatcher protein at room temperature for 2 hours, followed by a 70 μL reaction with SpyTag-DNA in a 2-molar excess relative to SpyCatcher protein. The reaction was carried out in the appropriate reaction buffer for SpyCatcher protein. Excess SpyTag-DNA was removed by purifying the protein using IMAC. The mixture was diluted 1:5 (final reaction volume = 350 μL) by adding 280 μL of binding buffer. Tris nickel-NTA (NiNTA) beads were prepared as follows: the supernatant was removed from 100 μL of bead slurry and the beads were washed three times with 300 μL of MilliQ water (centrifugation speed and time: 500 xg, 30 s). The beads were then equilibrated by two 300 μL exchanges with binding and washing buffers. The solution containing SpyTag-DNA and polymerase-SpyCatcher was added to the NiNTA beads and incubated on a rotating platform at 4°C for 2 hours. The supernatant was removed by centrifugation (500 xg, 30 s), and the beads were washed three times with 300 μL of washing buffer (centrifugation speed and time: 500 xg, 30 s) to remove excess SpyTag-DNA. Polymerase-DNA was recovered from the supernatant after incubation of NiNTA beads with 70 μL of elution buffer on a vortex mixer at 4°C for 2 hours. The sample buffer was then replaced with the buffer required for assembly onto the DNA origami scaffold using a desalted spinning SEC column, and sample purity was assessed by SDS-PAGE. In SDS-PAGE, proteins were visualized using the fluorescence signals of protein-specific dyes and covalently attached fluorophores.

[0231] DNA polymerase activity assay DNA polymerase function was assessed by measuring the extension of fluorescently labeled primers that bind to the target DNA strand. The reaction conditions are summarized below:

[0232] The reaction was terminated by adding 0.5 μl of 0.5 M EDTA, followed by denaturation at 95°C for 15 minutes. The extension of the DNA polymerase to the primers was visualized by non-denaturing (native) PAGE.

[0233] Non-denaturing PAGE 15% Tris-glycine non-denaturing polyacrylamide gel (containing 5% concentrated gel) was run at 150V for 10 min and at 250V for 45 min (until the dye reached 1.5 cm from the bottom).

[0234] Assembly of gold nanoparticles on DNA origami scaffolds DNA-coated gold nanoparticles were resuspended in IxTAE containing 6 mM MgCl2 and mixed with 500 pM purified DNA origami with single-stranded DNA extensions complementary to the thiolized DNA bound to the gold nanoparticles.

[0235] Assembly of DNA polymerase on a DNA origami scaffold 100 nM polymerase-DNA conjugate and 20 nM DNA origami scaffold were incubated in a 10 μL reaction volume for 2 hours at room temperature. The reaction buffer consisted of 10 mM Tris-HCl pH 8, 50 mM NaCl, and 10 mM MgCl2. Excess polymerase-DNA was removed by SEC as follows: • Prepare S300 columns, each complex containing 500 μL of slurry. • Centrifuge with 1000 g of resin for 2 min. • Wash the resin with 4 x 500 μL MQW for 1 min, 1000 g • Wash the resin with 3 x 500 μL imaging buffer for 1.5 min, 1000 g • Centrifuge the resin at 1000 g for 1.5 min. • Load approximately 10 μL of sample onto a 1x S300 column. • Centrifuge at 1000g for 4 min Nanodrop concentration in nM is the concentration in ng / μl divided by 4.8.

[0236] Surface plasmon resonance (SPR) Experiments were performed on a Biacore S200 instrument (GE Healthcare Life Sciences). All experiments were conducted at room temperature using Klenow reaction buffer at a flow rate of 10 μL / min. The CM3 sensor chip was coupled with streptavidin to near saturation (typically between 4000 and 7000 RU) using an amine coupling kit (GE Healthcare Life Sciences). After streptavidin coupling, biotinylated DNA strands were introduced into both the reference and experimental flow cells, and excess biotin binding sites were blocked with biotin in Klenow reaction buffer. Subsequently, DNA template strands were loaded into the experimental flow cell. Experiments were performed using two flow cells on the chip, with the remaining flow cell serving as a reference flow cell without DNA template. The surface was then conditioned with two Klenow reaction buffer injections, followed by the injection of a specified concentration of DNA polymerase. The figures show data after subtracting the reference values; all data were fitted using MATLAB 2020b.

[0237] Single-molecule total internal reflection fluorescence (TIRF) microscopy General Settings As described above, microfluidic devices and coverslips functionalized with BSA-biotin and streptavidin were prepared. 25 The sample was added to the imaging buffer of each channel at a concentration of 10 pM, and then washed with the imaging buffer. (This is similar to the approach used by McGuinness et al.) 26 Images were acquired on a custom-designed TIRF microscope with a power density of approximately 1–3 W cm⁻¹. -2 (Measured at the objective lens, the laser beam is perpendicular to the surface of the coverslip).

[0238] Single-particle photobleaching Single-particle photobleaching was performed using purified pre-assembled DNA origami scaffolds and DNA polymerase (fixed on coverslips in DNA origami imaging buffer, with a particle density of approximately 1000 particles per field of view (FOV). Images were acquired using excitation light at 488 nm (50 mW) and 647 nm (20 mW) with an exposure time of 200 ms, for 200 frames per FOV. The process was independently repeated three times, with 20 FOVs acquired each time. Images were analyzed using JIM-Immobilized-Microscopy-Suite (https: / / github.com / lilbutsa / JIM-Immobilized-Microscopy-Suite) to determine individual photobleaching steps and their corresponding step heights.

[0239] To measure fluorescence enhancement in the presence of metal nanoparticles, single-particle photobleaching was performed using a purified pre-assembled DNA origami scaffold with Alexa-647 fluorophores in the hotspot region. Figure 1 The scaffold was then incubated with 100 pM of DNA-coated 100 nm AuNP at a concentration of 100 pM in DNA origami imaging buffer for 2 hours, and then fixed onto a coverslip. Image acquisition was performed as described above.

[0240] Transient binding of fluorescent DNA strands to plasma nanoantennas To measure the fluorescence enhancement of multiple individual fluorophores over time, a docking strand containing a 7-base-pair ssDNA binding site (TCCTCCT)27 was incorporated into a DNA origami scaffold in the hotspot region. The DNA origami scaffold was purified, assembled with AuNP, and fixed onto a coverslip as described above. A 1 nM imaging strand (Alexa647-AGGAGGA) was then added, followed by 5000 frames of imaging with an exposure time of 200 ms and a 639 laser with a power of 20 mW.

[0241] Measuring the relationship between gold scattering intensity and polarization angle By using a polarizing filter and a zero-order half-wave plate in the excitation optical path, and rotating the half-wave plate in 10° increments, the polarization angle is changed in 20° increments, thus altering the incident polarization direction. Measurement results were obtained using a 1-second exposure time and a 20 mW 639 laser.

[0242] DNA sequencing measurements For sequencing measurements, as described above, the assembled nanoantennas were loaded onto a coverslip. The subsequent step involved adding 1 μM of biotinylated DNA complementary to the AuNP-coated DNA and holding for 1 min to immobilize the AuNPs on the coverslip, followed by washing with imaging buffer. Then, photobleaching measurements were performed as described to locate the correctly assembled particles. Sequencing measurements were then initiated using a 20 mW 639 and 568 laser with an exposure time of 100 ms and 20,000 frames acquired. Once the measurement was initiated, 100 nM dNTPs were infused into the channel at 50 μL / min for 2 min, then the flow rate was reduced to 10 μL / min.

[0243] Synthesis of fluorescently labeled dNTPs Material • Aminated dNTPs: γ-(6-aminohexyl)-dGTP, γ-(6-aminohexyl)-dATP, γ-(6-aminohexyl)-dCTP, γ-(6-aminohexyl)-dTTP, all in 50 μL (10 mM) solutions, stored at -20°C. • Alexa Fluor™ 647 NHS ester: Freshly prepared in DMSO, 25 nmol / tube, vacuum centrifuged and dried. • Alexa Fluor™ 568 NHS ester: Freshly prepared in DMSO, 25 nmol / tube, vacuum centrifuged and dried. • 10x reaction buffer: 1M NaHCO3 pH = 8.30 (freshly prepared) •HPLC buffer A: 0.1 M TEAA •HPLC buffer B: Acetonitrile Cross-linking reaction of amines and esters Alexa Fluor™ 568 / 647 NHS ester was dissolved in 5 μL DMSO to prepare a 5 mM stock solution. 10 μL of 0.4 mM Alexa Fluor™ 568 / 647 NHS ester and 1 mM γ-(6-aminohexyl)-dNTP were incubated in reaction buffer at room temperature for 2 hours, followed by purification by HPLC as described above.

[0244] Example 2. Design and Implementation of a Plasma Nanoantenna DNA Sequencing Instrument The plasma nanoantenna consists of three components. First, metallic nanoparticles (in this example, spherical gold nanoparticles with an average diameter of 100 nm). Second, a DNA polymerase. Third, a nanoscale scaffold constructed from DNA, which controls the spatial positioning of the metallic nanoparticles and the DNA polymerase, thereby directly generating an electromagnetic enhancement field at a single DNA polymerase located within the cavities between the gold nanoparticles. Figure 1 A). To enable sequencing readout, dNTPs are labeled with different colored fluorophores so that their sequential incorporation by DNA polymerase can be detected. This scaffold can also specifically fix plasmonic nanoantennas onto the surface of a glass coverslip for single-molecule fluorescence imaging. Figure 1 B).

[0245] The following sections describe the synthesis of each component and the assembly of all components into a complete nanoantenna DNA sequencer.

[0246] Design and synthesis of DNA origami scaffolds The design of a novel DNA sequencer requires placing DNA polymerase directly within a plasma-enhanced hotspot formed by two 100 nm AuNPs spaced 30 nm apart. Therefore, a novel three-dimensional DNA origami structure was designed, folded from M13mp18-derived scaffold chains and complementary staple chains.

[0247] DNA origami scaffold design The DNA origami scaffold consists of 86 parallel and interconnected DNA double helices, arranged in a U-shaped honeycomb lattice with a central cavity. Each helix between AuNPs is 7 turns or 72 bp (24.5 nm) in length. The cavity dimensions are approximately 25 x 12 x 24.5 nm. Figure 2A) should provide space for polymerase binding. For fixation on a coverslip for fluorescence microscopy imaging, the U-shaped cavity has a rigid tail approximately 80 nm long, consisting of four DNA helical bundles. The tail base has one or more biotin-modified DNA staple chains, capable of specifically fixing it to the BSA-biotin-coated coverslip via biotin-streptavidin interactions. Figure 2 (A and C). The overall shape of this DNA origami scaffold resembles a nano-wrench, with a U-shaped cavity resembling the head of a wrench and a tail resembling the handle. This DNA origami scaffold is called NanoSpanno (NS).

[0248] Single-stranded DNA strands can be designed to extend from the DNA helix to create specific binding sites (handles) for proteins or nanoparticles, which are then decorated with DNA strands (anti-handles) complementary to these handles. At the handle head, 10 DNA staples extend to the edges of both ends of the cavity, providing anchoring points for binding 100 nm AuNPs functionalized with DNA. Figure 2 (B and D). After the gold nanoparticles were bound to both sides of the cavity, the estimated gap length was approximately 35 nm. Within the cavity between these gaps, another staple extension with an orthogonal DNA sequence was placed as a handle for specifically binding a polymerase (6E) covalently bound to a complementary DNA strand, as detailed in Example 1. This allows the polymerase to be directly attached to the plasma-enhanced hotspot of the plasma nanoantenna (…). Figure 5 B).

[0249] The signal amplification achievable with NanoSpanno was calculated using finite element models by directly incorporating fluorescently labeled DNA staple chains (see below) into the plasma hotspots of NanoSpanno. These models showed that the fluorescent dye within the cavities between gold nanoparticles was observed to be more than 3000 times brighter than the fluorescent dye not located in the hotspots. Figure 3 ) Synthesis of DNA Origami Scaffolds Purified DNA origami samples were analyzed on agarose gel electrophoresis (AGE). Consistent with rigid, stable, and structurally homogeneous DNA origami, the NS migrated at a consistent rate, resulting in a single, clear band in the agarose gel electrophoresis. Furthermore, the migration rate was faster than that of the standalone M13mp18 scaffold, indicating a more compact structure and a smaller radius of gyration. Figure 3 A). These observations about AGE collectively indicate a well-structured DNA origami structure.

[0250] To verify that the DNA origami structure matched the design, we directly observed the purified structure using transmission electron microscopy. We observed NS structures with different orientations. Figure 3 Image B shows an example micrograph displaying multiple individual particles. Figure 3 C shows a two-dimensional classification average of multiple particles with different orientations. The DNA origami particles appear to have a well-consistent structure, with their shape and size matching the design, thus verifying their structural integrity.

[0251] Preparation of DNA-coated gold nanoparticles DNA hybridization facilitated the binding of metal nanoparticles to specific sites on the DNA origami scaffold. The single-stranded DNA on both sides of the cavity was designed to be complementary to the single-stranded DNA used for the chemical coating of gold nanoparticles with thiol-gold. 28 This article describes an optimized method for coating metal nanoparticles. Agarose gel electrophoresis verified the co-localization of thiolized DNA with gold nanoparticles. Figure 4 A), and under TEM, a halo was observed on the surface of the DNA-coated gold nanoparticles. This halo was not observed on uncoated gold nanoparticles. Figure 4 B). Combining these data, it is consistent with the effective coating of thiolated DNA with gold nanoparticles.

[0252] Because 100 nm gold nanoparticles readily aggregate, efficient methods for coating them are crucial. Extensive optimization is required to determine the correct length and sequence of these single-stranded DNAs, ensuring they meet the following specifications: 1. It will not cause the aggregation of DNA origami structures. 2. It can prevent the aggregation of gold nanoparticles. This is particularly challenging for nanoparticles with a diameter of 100 nm. 3. It can effectively and specifically couple paired nanoparticles to specific binding sites on DNA origami scaffolds. Table 2 below details the different DNA strands bound to the gold nanoparticles to test nanoparticle stability, DNA-gold binding efficiency, and assembly on NS DNA origami scaffolds.

[0253] Table 2. Detailed information on different DNA strands bound to gold nanoparticles

[0254] Nanoparticle stability After functionalizing AuNP with thiol-modified DNA, the precipitate was carefully resuspended in 1xTAE buffer containing 6 mM MgCl2 to ensure uniform sample dispersion. Stability was defined by the tendency to aggregate in 6 mM MgCl2, a concentration high enough to allow the gold nanoparticles to bind to DNA origami structures via DNA hybridization. Aggregation was detected by agarose gel electrophoresis, which produced distinctive DNA staining bands corresponding to the locations of the gold nanoparticles. Figure 5 And unique solution colors. Well-dispersed colloidal gold forms a clear red solution, while aggregated nanoparticles cause the solution to turn purple or clear, and aggregates are visible in test tubes. Figure 5 As shown in Table 2, sequences 5-8 (each 25 nucleotides in length) form a stable colloidal solution once they bind to gold nanoparticles, while sequences 1-3 cause the solution to turn purple or colorless, indicating that aggregation has occurred.

[0255] Design and synthesis of DNA polymerase Three polymerases were selected: DNA polymerase (Taq) from *Thermus aquaticus*, DNA polymerase from *Escherichia coli*, and DNA polymerase from *Escherichia coli*. Escherichia coli The study included a large (Klenow) fragment of DNA polymerase I and a DNA polymerase from bacteriophage Phi29 (Phi29). Each polymerase possesses different properties, which can be used for the development of single-molecule sequencers. Recombinant versions of these polymerases were designed to fuse with the SpyCatcher domain at their N-terminus via a glycine-serine-glycine (GSG) linker, enabling attachment to DNA origami scaffolds containing corresponding SpyTag-DNA "handles." An N-terminal polyhistidine tag (His-Tag) containing 6X histidine residues was also included for purification via IMAC. Figure 6 ) Purification of protein constructs The protein was purified by IMAC and SEC (see Example 1) and labeled with AlexFluor 647 (see Example 1) for visualization using fluorescence methods.

[0256] IMAC purification yielded a single elution peak. Figure 7 A), in which the fractions in SDS PAGE all show major bands at positions corresponding to the molecular weight of the target protein ( Figure 7 B). The peak fractions were combined and then further purified using SEC. The SEC chromatogram showed good monodispersity ( Figure 7 C), and the corresponding SDS-PAGE indicates a final purity >95% ( Figure 7D). The SEC chromatogram shows the Alexa 647 label, with a single elution peak visible in both A280 and A650 measurements. Figure 7 C). Corresponding SDS-PAGE analysis showed that the main band was visible in both the Alexa 647 channel and the stained bright field. Figure 7 D). The peak fractions were combined, and the absorbance at 280 nm and 650 nm was measured. The results show that the Alexa 647 labeling efficiency is 100%.

[0257] Synthesis of DNA-SpyTag peptide conjugates To achieve specific binding to the cavity of a DNA origami scaffold via DNA hybridization, the DNA strand needs to be covalently attached to a DNA polymerase. The inventors' method involves covalently attaching a SpyTag peptide (which forms an isopeptide bond with the SpyCatcher protein fused to the DNA polymerase) to the DNA strand. DNA-SpyTag conjugation is achieved using a bifunctional cross-linking agent (SMCC) composed of N-hydroxysuccinimide (NHS) ester and maleimide groups. These moieties are covalently bound, respectively, to the free amino groups chemically added to the target DNA strand and the thiol groups on the cysteine ​​side chains of the peptide. This produces the desired DNA-SpyTag construct (…). Figure 8 ).

[0258] Synthesis of maleimide DNA As described in Example 1 above, the synthesis of DNA-peptides involves a two-step process. First, maleimide DNA is synthesized by reacting SMCC with aminoated DNA. Figure 8 A), then purified by ethanol precipitation and HPLC. Maleimide can be selectively attached to the 5' or 3' end of the DNA strand. Figure 9 A and 9B show typical UV chromatograms of HPLC purification of 5'-modified and 3'-modified maleimide DNA, respectively. Figure 9 C shows a typical UV chromatogram of HPLC purification of 5' Cy5-labeled 3' maleimide DNA. In both cases, the main peak corresponds to maleimide DNA.

[0259] Synthesis of peptide-DNA conjugates In the second step, maleimide DNA is bound to the free thiol group on the cysteine ​​residue at the N-terminus of the target peptide and purified by HPLC (see Example 1). Figure 10 A and 10B show the UV chromatograms of DNA peptide conjugates with peptides linked to the 3' and 5' ends of DNA, respectively. Figure 10C shows the UV chromatogram of the peptide linked to the 3' end of 5'-Cy5-labeled DNA. In all cases, a single main peak corresponding to the DNA-peptide conjugate was present, indicating that the target DNA-peptide conjugate had high purity.

[0260] SpyTag-DNA conjugation with polymerase-SpyCatcher The SpyTag-DNA conjugate was bound to the polymerase-SpyCatcher fusion protein and purified using IMAC as described in Example 1. Conjugation efficiency was visualized by SDS-PAGE, showing that the DNA polymerase migration rate was significantly lower in the presence of SpyTag-DNA compared to DNA polymerase alone. Figure 11 -Klenow example). Regardless of whether the peptide binds to the 5' or 3' end of the DNA, a change in migration rate occurs ( Figure 11 By combining protein-specific staining and gel fluorescence imaging, fluorescently labeled DNA or protein was specifically located, demonstrating protein and DNA co-localization and confirming the successful assembly of DNA-labeled polymerase.

[0261] Verification of DNA polymerase function marked with DNA DNA polymerase function was assessed by measuring the extension of fluorescently labeled primers bound to the target DNA strand. The reaction was terminated at specific time points, and primer extension was visualized by non-denaturing PAGE. All polymerases appeared to successfully extend the fluorescent primers, resulting in slower band migration at 0.5–1 h compared to 0 h. Figure 16 A). Both 10 nM and 50 nM Klenow-SpyCatcher-SpyTag-DNA conjugates appeared to successfully extend the fluorescent primers, resulting in slower band migration at 24 h compared to 0 h. Figure 16 B). We conclude that DNA-tagged DNA polymerase retains its function.

[0262] Kinetic analysis of the interaction between polymerase and freely diffused primer-bearing DNA Determining the kinetics of the interaction between DNA polymerase and the primer-bearing target DNA strand is crucial to ensure that the experimental conditions we use can efficiently capture the target DNA molecule during the sequencing reaction. Since binding kinetics are affected by surface proximity (as in single-molecule TIRF microscopy), surface plasmon resonance (SPR) was used to measure the binding kinetics in bulk (Example 1).

[0263] For Klenow, combining the curve ( Figure 13A) is biphasic and extracts two concentration-dependent binding rate constants: a relatively fast rate constant (k_(a,fast) = 3.7 ± 0.3 × 10^6 mol^(-l) s^(-l)) and a slower rate constant (k_(a,slow) = 7.4 ± 0.3 × 10^4 mol^(-l) s^(-l)). Dissociation curves ( Figure 13 B) is also biphasic, with a fast dissociation rate constant (k_(d,fast) = 1.8 ± 1.1 × 10^(-2) s^(-1)) and an average dissociation time of 38 seconds, and a slow dissociation rate constant (k_(d,slow) = 5.9 ± 1.1 × 10^(-4) s^(-1)) and an average dissociation time of 20 minutes. The dissociation rate is determined by the proportion of polymerase bound under steady-state conditions (K_D = 26 ± 3 nM, Figure 13 C).

[0264] For Phi29, combined with the curve ( Figure 13 D) is also biphasic, and two concentration-dependent binding rate constants were extracted. One relatively fast rate constant (k_(a,fast) = 1.1 ± 0.3 × 10^6 mol^(-l) s^(-l)) and one slower rate constant (k_(a,slow) = 2.1 ± 0.3 × 10^5 mol^(-l) s^(-l)). Dissociation curves ( Figure 13 E) is also biphasic, with a fast dissociation rate constant (k_(d,fast) = 1.4 ± 0.1 × 10^(-2) s^(-1)) and an average dissociation time of 50 seconds; and a slower dissociation rate constant (k_(d,slow) = 1.2 ± 0.2 × 10^(-3) s^(-1)) and an average dissociation time of 10 minutes. The dissociation constant is determined by the proportion of polymerase bound under steady state (K_D = 56 ± 9 nM, Figure 13 F).

[0265] Assembly of Plasma Nanoantennas A major challenge was determining the sequence and length of the DNA strands that could stably immobilize the gold nanoparticles to prevent their aggregation in 6 mM MgCl2 (essential for promoting the binding of gold nanoparticles to the DNA origami scaffold via DNA hybridization) without causing the DNA origami scaffolds themselves to aggregate. Various combinations of DNA strand and nanoparticle sizes were tested. These combinations and their efficiency in assembling target structures are summarized in Table 3 below.

[0266] Table 3. Summary of gold nanoparticle sizes and DNA sequences encapsulating these nanoparticles assembled on DNA origami scaffolds.

[0267] Evaluating the assembled plasma nanoantennas using electron microscopy The relative DNA origami scaffold was tested with different molar excesses of gold nanoparticles and different binding site configurations (0, 1, or 2 binding sites) of the gold nanoparticles. After incubation, the sample turned red, indicating the absence of a large number of unwanted aggregates in the solution. Figure 14 A).

[0268] The samples were further analyzed by agarose gel electrophoresis. Individual DNA origami (constructed with 0, 1, or 2 binding sites) produced a single major band as expected. In the presence of excess gold nanoparticles, this band was not observed in DNA origami configurations with 1 or 2 gold binding sites. Importantly, when the DNA origami was constructed without binding sites, a band corresponding to the individual DNA origami could still be observed in the presence of excess gold nanoparticles. Figure 14 A). In summary, this indicates that gold nanoparticles can only specifically bind to DNA origami in the presence of gold binding sites.

[0269] Individual gold nanoparticles produce ladder-like bands, indicating oligomerization. However, no additional bands were observed when the gold nanoparticles bound to the DNA origami scaffold, making it difficult to determine the number of gold nanoparticles bound to the DNA origami scaffold. Figure 14 A).

[0270] To quantify the number of gold nanoparticles bound to the DNA origami scaffold and to confirm that they have bound to their specific binding sites, the DNA origami-gold complex was visualized using transmission electron microscopy. Figure 14 B shows an example of correctly assembled particles observed in an electron micrograph, where two gold nanoparticles are bonded to either side of the DNA origami scaffold, consistent with the design. In principle, this configuration of gold nanoparticles and DNA origami constitutes a functional plasmonic nanoantenna that will enhance the fluorescence intensity of fluorescent molecules within the cavity of the DNA origami scaffold. Figure 14 C shows examples of nanoparticle monomers and aggregates contained in electron micrographs. Some aggregates appear to be caused by daisy chain linkages mediated by DNA origami, while others appear to be the result of direct interactions between gold nanoparticles.

[0271] Assembly yield of plasma nanoantenna particles was evaluated using electron microscopy. Transmission electron microscopy was used to quantitatively analyze the proportion and configuration (monomer, dimer, aggregate) of gold nanoparticles bound to the DNA origami scaffold. The DNA origami scaffold had different configurations (0, 1 or 2 binding sites) and two different thiol DNA extensions (25-TG and 25-AC).

[0272] With two binding sites, 100% of the DNA origami nanospanno structures bound to AuNP, and no free DNA origami structures were observed in electron microscopy. 22% of the molecules were dimers, approximately 10% were monomers, and approximately 65% ​​were aggregates or other poorly assembled structures. Figure 19 (A and D). For DNA origami structures with a single binding site, only half bind to AuNP. No dimer assembly was observed; 14–22% of AuNPs bound to individual DNA origami structures. Figure 19 B and E). For DNA origami structures without gold-binding sites, EM only showed free gold and free NS, with no evidence of assembled structure (B and E). Figure 19 (C and F). These data combined indicate that a specific and desired plasmonic nanoantenna structure was successfully assembled, consisting of two 100 nm gold nanoparticles located on either side of a cavity capable of immobilizing DNA polymerase.

[0273] Colocalization of DNA polymerase and DNA origami scaffold In this section, we describe the specific attachment of polymerase-DNA to the cavity of a DNA origami scaffold. This is achieved through extended hybridization of the polymerase-bound DNA with complementary DNA staples located within the cavity. Figure 16 ).

[0274] Measurement of global colocalization using agarose gel electrophoresis Overall binding was assessed by observing the colocalization of DNA origami scaffolds labeled with Alexa 488 fluorescent dye and DNA polymerase labeled with Alexa 647 dye on agarose gel electrophoresis. Colocalization of DNA polymerase and DNA origami was only observed when the DNA polymerase bound to SpyTagDNA. Furthermore, we observed that colocalization led to a decrease in the migration rate of both the individual DNA origami and the individual DNA polymerase. Figure 17 These data indicate that DNA polymerase binds through specific hybridization with DNA staple extensions within the DNA origami cavity.

[0275] Quantification of colocalization yield using single-molecule fluorescence microscopy To quantify the co-localization yield of DNA polymerase and DNA origami scaffolds, single-molecule fluorescence microscopy was used to determine the proportion of individual DNA origami molecules bound to a single DNA polymerase. The presence of individual DNA polymerases was determined by photobleaching, with yield analysis performed using only molecules exhibiting a single photobleaching step. Figure 18(A and B). In contrast, we found that the Alexa 488 fluorophore bound to the DNA origami was resistant to photobleaching. Therefore, we determined the presence of individual DNA origami particles by the initial fluorescence intensity upon excitation with 488 nm light. By plotting the initial intensity distribution of all fluorescent particles, it was easy to distinguish individual DNA polymerases from oligomers or aggregates (A and B). Figure 18 C). Through these measurements, we were able to determine that the yield of single DNA origami scaffolds co-localized with a single DNA polymerase molecule was approximately 90%, independent of the location of the DNA polymerase binding site. Figure 18 D). We noted that, as a positive control, we annealed excess Alexa 647 DNA complementary to the DNA polymerase binding site. This provides a measurement of the maximum possible occupancy of the DNA polymerase, also 90% ( Figure 18 D).

[0276] Example 3. Characterization of Plasma Nanoantennas Fluorescent dyes that enhance fluorescence binding in hot spots To determine the function of the plasmonic nanoantenna, we experimentally quantified the intensity of the fluorophores bound to the DNA origami scaffold (either alone or with a single or a pair of bound gold nanoparticles).

[0277] Intensity distribution measured by single-molecule photobleaching The intensity of Alexa 647 fluorophores immobilized within plasma nanoantennas was measured using single-molecule photobleaching assays. Alexa 647-tagged DNA origami scaffolds with zero or two AuNP binding sites were incubated with 100 pM of 500 pM of AuNP for 2 hours, then diluted 1:10 for TIRF microscopy measurements (see Example 1, titled "..."). Single-molecule total Internal reflection fluorescence (TIRF) microscopy (the part marked ""). Figure 19 A provides an example of a single photobleaching step on a DNA origami scaffold without AuNPs (blue), whose intensity is significantly lower than that of a single fluorophore in a DNA origami scaffold containing a pair of AuNPs (red). Figure 19 B shows the intensity distribution of approximately 3000 independent assemblies with or without AuNP binding sites (blue) or with two AuNP binding sites (red). These data indicate a broad distribution of fluorescence enhancement ranging from 1 to 200 times in the presence of two gold nanoparticles. This broad distribution is due to a variety of factors, including the assembly yield of the 2xAuNP:1xDNA origami scaffold, the orientation of the plasmonic nanoantennas on the coverslip, the possible anisotropy of the fluorophore labeling, variations in the distance between nanoparticle pairs, and the inhomogeneity of nanoparticle size and shape. Bleaching rate ( Figure 19C) is similar, indicating that the total photon count of the fluorophore is higher in the presence of AuNP.

[0278] TEM experiments showed that the yield of correctly assembled nanoantennas was 22%. Due to the significant scattering of gold nanoparticles, and the fact that their scattering cross-section at 488 nm is proportional to the number of particles in the cluster, single DNA origami scaffolds with 0, 1, 2, or more gold nanoparticles were identified. Figure 19 D). Importantly, the proportion of dimerized gold nanoparticles is similar to that observed directly by electron microscopy (D). Figure 18 (D and Table 4). The intensity distribution of fluorophores in DNA origami scaffolds with 0, 1, or 2 gold nanoparticles, as measured by scattering, was differentiated. The average fluorophore intensity on DNA scaffolds with two gold nanoparticles was 10 times higher than that on DNA scaffolds without gold nanoparticles. Figure 19 E). Notably, the range of fluorescence enhancement varied considerably when two gold nanoparticles were present; some particles showed no fluorescence enhancement, while others exhibited enhancements exceeding 100-fold. This is not surprising, as the plasmon enhancement effect depends on the alignment of the gold nanoparticle pair with the polarization direction of the incident light. Quantification of the photobleaching rate again revealed little correlation with the number of bound gold nanoparticles (E). Figure 19 F).

[0279] Table 4. Population quantification of origami bleaching steps with individual Alexa647 markers having corresponding scattering intensities of 0, 1, or 2 AuNPs.

[0280]

[0281] Enhanced fluorescence intensity as measured by DNA PAINT While photobleaching experiments can be used to determine the assembly composition, stoichiometry, and fluorescence intensity of static molecules, DNA sequencing requires repeated measurements of fluorescent dye transiently bound to hot spots within plasmonic nanoantennas. To establish the conditions required for DNA sequencing, repeated transient binding of fluorescent DNA strands to 7-base DNA staple extensions within the plasmonic nanoantenna hot spots was measured. Repeated measurements of the same molecule also revealed whether bright particles remained consistently bright or whether there were significant fluctuations in the fluorescence intensity of individual plasmonic nanoantennas.

[0282] Figure 20The results show fluorescence intensity spikes caused by the transient binding of fluorescent DNA strands to the cavities of the DNA origami structure, both in the presence and absence of fixed gold nanoparticle pairs. In the presence of gold pairs, a 10-50 fold increase in fluorescence was observed, confirming the function of the plasmonic nanoantenna in enhancing the fluorescence signal. The large range of enhanced intensity is also likely due to fluctuations in the orientation of the gold nanoparticles during the experiment. A few intensity spikes were also observed in the absence of imaging strands. Figure 21 These spikes are thought to originate from fluctuations in the orientation of gold nanoparticles, which increase scattering intensity and manifest as peaks in the intensity trace, potentially leading to false positives in sequencing measurements.

[0283] The effect of the rotating polarization field on the orientation of gold nanoparticles is characterized by the following experiment.

[0284] Enhanced fluorescence of DNA polymerase in hotspots Next, to complete the synthesis of the plasma nanoantenna DNA sequencer, fluorescently labeled DNA polymerase molecules were bound to the hot spots of the plasma nanoantenna. The intensity of individual fluorescent DNA polymerases (without gold nanoparticles and pairs containing gold nanoparticles) bound to the DNA origami scaffold cavity was measured using single-molecule photobleaching measurements. Figure 22 The left and right sides respectively show example bleaching traces with and without gold, demonstrating that the fluorescence signal is enhanced by two orders of magnitude in the presence of gold pairs. As mentioned above, the plasmonic enhancement with gold nanoparticle pairs exhibits a wider range of distributions due to the rotational freedom of the gold spheres, consistent with expectations. Nevertheless, these data directly demonstrate that DNA polymerase has been successfully immobilized in the hotspot of the plasmonic nanoantenna. This is the first instance of fluorescence enhancement of protein molecules achieved via plasmonic nanoantennas.

[0285] Characterizing fluorescence enhancement at different polarization angles To determine the effect of incident light polarization on the scattering intensity of the plasma nanoantenna, simulations were performed on the scattering cross-section, light intensity, and fluorescence enhancement of the nanoantenna oriented perpendicular to the glass surface under different incident polarization angles. Figure 23 AI shows how the TIR intensity above the coverslip changes from fully p-polarized (parallel, 0°, polarization direction perpendicular to the coverslip) to fully s-polarized (perpendicular, 90°, polarization direction along the coverslip direction). This was used for ( Figure 23 Aii) When the incident polarization is changed by a half-wave plate, the polarization angle is experimentally calibrated based on the measured background intensity (see Example 1, titled "..."). Measuring the relationship between gold scattering intensity and polarization angle "part). Figure 23Bi shows the simulated scattering cross-sections of the nanoantenna oriented along the TIRF field propagation direction (blue) or perpendicular to the propagation direction (black). In the blue orientation, the nanoantenna is not optimally aligned with the incident polarized light, resulting in a lower average scattering cross-section across all polarization angles, and its variation with polarization angle follows the variation of TIR intensity. In the black orientation, because the s-polarized light is aligned with the nanoantenna in this orientation, the scattering cross-section exhibits a 90° phase difference and a higher average value. Figure 27 Bii shows the intensity of scattered light from various particles as the polarization angle changes, which can be measured experimentally to measure nanoantennas aligned perpendicular to the TIRF field propagation direction (black), parallel to the propagation direction (blue), or a single AuNP (green).

[0286] When the TIRF field is s-polarized, the measured intensity varies significantly depending on the orientation of the nanoantenna. To prevent this during sequencing experiments, AuNPs are immobilized on glass coverslips via biotin:streptavidin interactions. This is achieved by initially attaching AuNPs to a functionalized coverslip using a biotinylated DNA origami scaffold, followed by the inflow of biotinylated DNA complementary to the AuNP-bound DNA. Measurements of the particles were taken approximately 30 minutes after this step, showing a significant reduction in the number of intensity spikes. Figure 24 ).

[0287] The effect of nanoantenna orientation on the enhancement of Alexa-647 fluorophores was also simulated. Figure 25 The results showed that the enhancement variation was less than 1 to 200 times. The enhancement distribution of randomly oriented nanoantenna groups was similar to that seen above.

[0288] Example 4. Single-molecule DNA sequencing reaction fluorescently labeled dNTPs DNA sequencing is achieved by detecting the incorporation of individual fluorescent nucleotides by DNA polymerase located at a plasma hotspot. Each nucleotide (ATP, GTP, TTP, or CTP) is labeled with a different colored dye. Importantly, the fluorescent probe needs to be attached to the terminal phosphate group of the nucleotide to prevent polymerase reaction termination and to ensure that the fluorescent probe is released during phosphodiester bond formation when the nucleotide is incorporated. Nucleotides with a 6-aminohexyl group attached to the distal phosphate group were purchased from Jena Biosciences and can be used to attach succinimide fluorophores using the NHS esterification reaction chemistry (Sigma), followed by purification by HPLC.

[0289] Aminated dGTP typically elutes at approximately 12 min, while Alexa647-labeled dGTP typically elutes at approximately 32 min. Figure 26A). Evaporate the fraction containing the pure product, then co-evaporate with methanol:MQW = 1:1 (2×). Dissolve the residue in water and quantify by UV-Vis spectrophotometry, then aliquot into 500 pmol / tubes (so that it can be resuspended in 10 μL MQW to obtain a 50 μM working stock solution), dry and store at -20°C.

[0290] Alexa568 has two isomers, which typically elute at approximately 41 and 42 minutes, and are blue at both 260 nm and 578 nm. Figure 26 B). These isomers have hydrolysis byproducts that elute at 30 and 35 minutes. Aminated dGTP typically elutes at about 12 minutes and appears orange at 260 nm, while Alexa568-labeled dGTP, due to its two isomers, typically elutes at about 31 and 36 minutes and appears orange at both 260 nm and 578 nm. Figure 26 B). Aminated dATP typically eluted at approximately 15 minutes, while Alexa568-labeled dTTP typically eluted at approximately 32 and 36 minutes. Aminated dTTP typically eluted at approximately 10 minutes, while Alexa568-labeled dTTP typically eluted at approximately 32 and 36 minutes. Finally, aminated dCTP typically eluted at approximately 5 minutes, while Alexa568-labeled dCTP typically eluted at approximately 32 and 36 minutes. Figure 26 C).

[0291] Constructing Plasma Nanoantennas for DNA Sequencing To conduct preliminary proof-of-concept DNA sequencing experiments, a plasma nanoantenna was constructed comprising a single DNA polymerase with a 71-base-fixed single-stranded DNA adapter and a primer-bearing target DNA strand. Figure 27 This configuration eliminates the need to capture freely diffusing DNA. Therefore, the number of parameters in the experiment is minimized. The length of DNA reads will also be limited by steric hindrance.

[0292] Two-color DNA sequencing experiments were performed using Alexa 647-labeled dGTP and Alexa 568-labeled dATP. As a proof of concept, according to Figure 27The illustrated configuration assembled a plasmonic nanoantenna DNA sequencer and fixed it to the surface of a glass coverslip for imaging using a TIRF microscope. To identify the fully assembled plasmonic nanoantenna sequencer, single-molecule photobleaching assays were performed and scattering intensity was measured to identify particles containing exactly one DNA origami scaffold, one DNA polymerase, and a pair of gold nanoparticles. Simultaneous excitation with 568 and 647 Å lasers was used to monitor the change in fluorescence intensity over time around these fully assembled particles to produce a two-color intensity trace. This resulted in a significant increase in fluorescence background as expected. Furthermore, distinct fluorescence spikes significantly above the background were observed (…). Figure 28 Base identification is simply defined as any intensity spike that is more than 3 standard deviations above the background. This base identification algorithm enables multi-plasma nanoantenna DNA sequencers to identify completely correct sequences of 8–16 base read lengths. Figure 28 Two example traces are shown. Notably, in the control experiment lacking a single key component, such as DNA polymerase or the target DNA template strand, the correct sequence was not identified.

Claims

1. A method for sequencing nucleic acid analytes, the method comprising: (I) Under certain time and conditions, a nucleic acid polymerase is brought into contact with the nucleic acid analyte and the labeled nucleotide, such that the labeled nucleotide is sequentially incorporated by the nucleic acid polymerase into a polynucleotide having a sequence complementary to the polynucleotide sequence of the nucleic acid analyte, wherein the nucleic acid polymerase is located in an electric field enhanced region; And each labeled nucleotide includes: (i) Adenine nucleotide (A), guanine nucleotide (G), thymine nucleotide (T) or cytosine nucleotide (C). (ii) Fluorescein, and (iii) A polyphosphate linker that binds the nucleotide to the fluorophore; In this configuration, A, G, T, and C are each independently linked to the fluorophore via the polyphosphate linker. When the fluorophore linked to the corresponding nucleotide is excited, each of the labeled nucleotides A, G, T, and C exhibits a unique fluorescence emission characteristic, and The unique fluorescence emission characteristics of the labeled nucleotide are enhanced when incorporated by the nucleic acid polymerase into a sequence complementary to the polynucleotide sequence of the nucleic acid analyte; and (II) Detecting the sequence of enhanced unique fluorescence emission features when the labeled nucleotide is sequentially incorporated by the nucleic acid polymerase into the polynucleotide having a sequence complementary to the polynucleotide sequence of the nucleic acid analyte, thereby determining the sequence of the nucleic acid analyte by determining the sequence of the nucleotides incorporated into the polynucleotide having a sequence complementary to the polynucleotide sequence of the nucleic acid analyte.

2. The method of claim 1, wherein the unique fluorescence emission characteristics of the labeled nucleotide are not enhanced outside the electric field enhancement region.

3. The method according to claim 1 or 2, wherein the enhanced unique fluorescence emission feature of the labeled nucleotide is increased by two times or more compared to the unique fluorescence emission feature of the corresponding labeled nucleotide outside the enhanced electric field region.

4. The method of claim 1, wherein the enhanced unique fluorescence emission feature of the labeled nucleotide is enhanced by an order of magnitude or more compared to the unique fluorescence emission feature of the corresponding labeled nucleotide outside the enhanced electric field region.

5. The method according to any one of claims 1 to 4, wherein the electric field enhancement region is generated by a plasma hot spot, and wherein the plasma hot spot is generated by a plasma nanoantenna.

6. The method of claim 5, wherein the plasma nanoantenna comprises at least two plasma nanoparticles.

7. The method of claim 6, wherein each plasma nanoantenna comprises: Two plasma nanoparticles; Nanoscale nucleic acid scaffolds; and The nucleic acid polymerase; in: (a) The two plasma nanoparticles are attached to the nanoscale nucleic acid scaffold and positioned relative to each other, such that there is a plasma hotspot between the two plasma nanoparticles. (b) The plasma hotspot includes a region not occupied by the nanoscale nucleic acid scaffold, and (c) The nucleic acid polymerase binds to the nanoscale nucleic acid scaffold and is located in the region not occupied by the nanoscale nucleic acid scaffold, and (d) Optionally, the nucleic acid analyte is bound to the nanoscale nucleic acid scaffold.

8. The method of claim 7, wherein the region not occupied by the nanoscale nucleic acid scaffold is defined by a three-dimensional space having a length (L) between the two plasma nanoparticles, a height (H) perpendicular to (L) at the midpoint (L / 2), and a width (W) perpendicular to L and H at L / 2, 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 plasma nanoparticles.

9. The method of claim 8, wherein L is selected from 20 nm to 100 nm.

10. The method of claim 9, wherein L is selected from 20 nm to 50 nm.

11. The method according to any one of claims 7 to 10, wherein the volume of the region not occupied by the nanoscale nucleic acid scaffold is at least about 1 zL, preferably about 10 zL to about 50 zL.

12. The method according to any one of claims 7 to 10, wherein the region not occupied by the nanoscale nucleic acid scaffold is 80% or more of the plasma hotspot.

13. The method of claim 12, wherein the region not occupied by the nanoscale nucleic acid scaffold is 90% or more of the plasma hotspot.

14. The method according to any one of claims 7 to 13, wherein the region not occupied by the nanoscale nucleic acid scaffold is amorphous.

15. The method according to any one of claims 7 to 13, wherein the region not occupied by the nanoscale nucleic acid scaffold is spherical or cubic.

16. The method according to any one of claims 7 to 15, wherein the plasma nanoantenna comprises the nucleic acid analyte bound to the nanoscale nucleic acid scaffold.

17. The method of claim 16, wherein the nucleic acid analyte is non-covalently bound to the nanoscale nucleic acid scaffold.

18. The method of claim 17, wherein the nucleic acid analyte is non-covalently bound to the nanoscale nucleic acid scaffold via hydrogen bonds.

19. The method according to any one of claims 7 to 18, wherein each of the two plasma nanoparticles is independently selected from metal nanoparticles.

20. The method of claim 19, wherein each of the two plasma nanoparticles is independently selected from gold, silver, aluminum, copper, palladium, and platinum nanoparticles or alloys thereof.

21. The method of claim 20, wherein each of the two plasma nanoparticles is a gold nanoparticle.

22. The method according to any one of claims 7 to 21, wherein the maximum diameter of each of the two plasma nanoparticles is about 5 nm to about 500 nm.

23. The method of claim 22, wherein the maximum diameter of each of the two plasma nanoparticles is about 50 nm to about 150 nm.

24. The method of claim 23, wherein the maximum diameter of each of the two plasma nanoparticles is about 80 nm to 120 nm.

25. The method according to any one of claims 7 to 24, wherein each of the two plasma nanoparticles is coated with a coating layer.

26. The method of claim 25, wherein the coating layer substantially covers the surface of each of the two plasma nanoparticles.

27. The method of claim 25 or 26, wherein the coating layer reduces the aggregation of the two plasma nanoparticles by 50% or more relative to the two plasma nanoparticles without the coating layer.

28. The method according to any one of claims 25 to 27, wherein the coating layer comprises a plurality of oligonucleotides.

29. The method of claim 28, wherein each of the plurality of oligonucleotides comprises about 20 nucleotides to about 50 nucleotides.

30. The method of claim 29, wherein each of the plurality of oligonucleotides comprises about 20 nucleotides to about 30 nucleotides.

31. The method according to any one of claims 28 to 30, wherein each nucleotide of each of the plurality of oligonucleotides is selected from pyrimidine nucleotides.

32. The method according to any one of claims 6 to 31, wherein the nanoscale nucleic acid scaffold is substantially composed of double-stranded DNA, preferably composed of parallel interconnected double helical strands.

33. The method according to any one of claims 7 to 31, wherein the nanoscale nucleic acid scaffold is composed of a DNA origami structure.

34. The method of claim 33, wherein at least a portion of the DNA origami structure is assembled into a U-shaped or curved structure.

35. The method according to any one of claims 7 to 34, wherein the nanoscale nucleic acid scaffold comprises a first face to which a first of the two plasma nanoparticles is anchored, and a second face to which a second of the two plasma nanoparticles is anchored.

36. The method of claim 35, wherein the first surface of the scaffold and the second surface of the scaffold are located on the same side or adjacent side of the nanoscale nucleic acid scaffold.

37. The method of claim 35, wherein the first surface of the scaffold and the second surface of the scaffold are located on opposite sides of the nanoscale nucleic acid scaffold.

38. The method according to any one of claims 35 to 37, wherein the two plasma nanoparticles are respectively anchored to the first surface and the second surface of the scaffold via one or more nucleic acid adapters. Each nucleic acid linker is formed by an oligonucleotide coated on the surface of the plasma nanoparticles, and the oligonucleotide hybridizes with a polynucleotide forming part of or extending from the nanoscale nucleic acid scaffold. The sequences of the oligonucleotides and polynucleotides capable of hybridization are complementary or substantially complementary to each other.

39. The method according to any one of claims 35 to 38, wherein the nanoscale nucleic acid scaffold comprises one or more polynucleotides extending from the first side of the scaffold, each polynucleotide comprising a sequence complementary to the sequence of one or more oligonucleotides coating one of the two plasma nanoparticles.

40. The method of any one of claims 35 to 39, wherein the nanoscale nucleic acid scaffold comprises one or more polynucleotides extending from the second side of the scaffold, each polynucleotide comprising a sequence complementary to the sequence of one or more oligonucleotides coating one of the two plasma nanoparticles.

41. The method of claim 39 or 40, wherein the one or more polynucleotides extending from the surface of the scaffold are independently selected from single-stranded DNA.

42. The method according to any one of claims 7 to 41, wherein the nucleic acid polymerase is bound to or immobilized on the nanoscale nucleic acid scaffold via a nucleic acid adapter.

43. The method of claim 42, wherein the nucleic acid adapter is double-stranded and comprises: (i) a polynucleotide covalently or non-covalently bound to the nucleic acid polymerase via amino acids within the nucleic acid polymerase, and (ii) a polynucleotide forming part of or extending from the nanoscale nucleic acid scaffold, wherein the polynucleotides at (i) and (ii) comprise complementary or substantially complementary single-stranded DNA sequences capable of hybridizing with each other.

44. The method of claim 43, wherein the polynucleotide at (i) comprises a peptide tag, the peptide tag being bound to the nucleic acid polymerase via a heteropeptide bond formed between an amino acid within the nucleic acid polymerase and an amino acid within the peptide tag.

45. The method according to claim 42 or 43, wherein the nucleic acid adapter binds to the nucleic acid polymerase via an amino acid within the N-terminal domain of the nucleic acid polymerase.

46. ​​The method according to any one of claims 1 to 45, wherein the nucleic acid polymerase is a DNA polymerase.

47. The method according to any one of claims 1 to 46, wherein the nucleotide of the labeled nucleotide is further selected from synthetic nucleotides.

48. The method of claim 47, wherein the synthetic nucleotide is selected from 5-methylcytidine, N-methylcytidine, and N6-methyladenosine.

49. The method according to claim 47 or 48, wherein the synthetic nucleotide is linked to a fluorophore via the polyphosphate, and wherein each of the nucleotides A, G, T, C and the synthetic nucleotide has a unique fluorescence emission characteristic when the fluorophore linked to the respective nucleotide is excited.

50. The method according to any one of claims 1 to 49, wherein the unique fluorescence emission characteristics are distinguishable from each other based on differences in peak emission wavelength, differences in fluorescence emission intensity, differences in fluorescence emission duration, differences in duration between consecutive fluorescence emissions, or any combination thereof.

51. The method according to any one of claims 1 to 50, wherein each different type of nucleotide is linked to a fluorophore having a unique peak emission wavelength.

52. The method of claim 51, wherein the unique peak emission wavelengths are spaced 10 nm or more apart from each other.

53. The method according to any one of claims 1 to 50, wherein two or more types of nucleotides are attached to the same fluorophore, and different types of nucleotides are distinguished based on differences in fluorescence emission intensity and / or fluorescence emission duration.

54. The method according to any one of claims 1 to 53, wherein the polyphosphate is triphosphate, tetraphosphate, pentaphosphate or hexaphosphate.

55. The method of claim 54, wherein the fluorophore binds to the phosphate furthest from the nucleotide.

56. The method according to any one of claims 1 to 55, wherein the sequence of enhanced unique fluorescence emission features at (II) is detected using a fluorescence microscope or fluorometer.

57. The method according to any one of claims 1 to 56, wherein the sequence of enhanced unique fluorescence emission features at (II) is detected using total internal reflection fluorescence (TIRF) microscopy.

58. The method according to any one of claims 1 to 57, wherein the nucleic acid analyte is DNA.

59. The method according to any one of claims 1 to 57, wherein the nucleic acid analyte is complementary DNA (cDNA) derived from an RNA sample.

60. The method of claim 5, or the method of any one of claims 6 to 59 when claims 6 to 59 are dependent on claim 5, wherein the method includes using a plasma nanoantenna array.

61. A plasma nanoantenna, said plasma nanoantenna comprising: Two plasma nanoparticles; Nanoscale nucleic acid scaffolds; and Nucleic acid polymerase; in: (a) The two plasma nanoparticles are attached to the nanoscale nucleic acid scaffold and positioned relative to each other, such that there is a plasma hotspot between the two plasma nanoparticles. (b) The plasma hotspot includes a region not occupied by the nanoscale nucleic acid scaffold, and (c) The nucleic acid polymerase binds to the nanoscale nucleic acid scaffold and is located in the region not occupied by the nanoscale nucleic acid scaffold, and (d) Optionally, the nucleic acid analyte is bound to the nanoscale nucleic acid scaffold.

62. The plasma nanoantenna of claim 61, wherein the region not occupied by the nanoscale nucleic acid scaffold is defined by a three-dimensional space having a length (L) between the two plasma nanoparticles, a height (H) perpendicular to (L) at the midpoint (L / 2), and a width (W) perpendicular to L and H at L / 2, 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 plasma nanoparticles.

63. The plasma nanoantenna of claim 62, wherein L is selected from 20 nm to 100 nm.

64. The plasma nanoantenna of claim 63, wherein L is selected from 20 nm to 50 nm.

65. The plasma nanoantenna according to any one of claims 61 to 64, wherein the volume of the region not occupied by the nanoscale nucleic acid scaffold is at least about 1 zL, preferably about 10 zL to about 50 zL.

66. The plasma nanoantenna according to any one of claims 61 to 65, wherein the region not occupied by the nanoscale nucleic acid scaffold is 80% or more of the plasma hot spot.

67. The plasma nanoantenna of claim 66, wherein the region not occupied by the nanoscale nucleic acid scaffold is 90% or more of the plasma hot spot.

68. The plasma nanoantenna according to any one of claims 61 to 67, wherein the region not occupied by the nanoscale nucleic acid scaffold is amorphous.

69. The plasma nanoantenna according to any one of claims 61 to 67, wherein the region not occupied by the nanoscale nucleic acid scaffold is spherical or cubic.

70. The plasma nanoantenna according to any one of claims 61 to 69, wherein the plasma nanoantenna comprises a polynucleotide forming part of or extending from the nanoscale nucleic acid scaffold, the polynucleotide being capable of hybridizing with another polynucleotide contained in or linked to a target nucleic acid analyte, such that when the corresponding polynucleotide hybridizes, the nucleic acid analyte preferably non-covalently binds to the nanoscale nucleic acid scaffold via hydrogen bonds.

71. The plasma nanoantenna of claim 70, wherein the polynucleotides capable of hybridizing with each other comprise sequences that are complementary or substantially complementary to each other.

72. The plasma nanoantenna according to any one of claims 61 to 71, wherein the plasma nanoantenna comprises a nucleic acid analyte bound to the nanoscale nucleic acid scaffold.

73. The plasma nanoantenna according to any one of claims 61 to 72, wherein each of the two plasma nanoparticles is independently selected from metal nanoparticles.

74. The plasma nanoantenna of claim 73, wherein each of the two plasma nanoparticles is independently selected from gold, silver, aluminum, copper, palladium and platinum nanoparticles or alloys thereof.

75. The plasma nanoantenna of claim 74, wherein each of the two plasma nanoparticles is a gold nanoparticle.

76. The plasma nanoantenna according to any one of claims 61 to 75, wherein the maximum diameter of each of the two plasma nanoparticles is about 5 nm to about 500 nm.

77. The plasma nanoantenna of claim 76, wherein the maximum diameter of each of the two plasma nanoparticles is about 50 nm to about 150 nm.

78. The plasma nanoantenna of claim 77, wherein the maximum diameter of each of the two plasma nanoparticles is about 80 nm to 120 nm.

79. The plasma nanoantenna according to any one of claims 61 to 78, wherein each of the two plasma nanoparticles is coated with a coating layer.

80. The plasma nanoantenna of claim 79, wherein the cladding layer substantially covers the surface of each of the two plasma nanoparticles.

81. The plasma nanoantenna of claim 80, wherein the coating layer reduces the aggregation of the two plasma nanoparticles by 50% or more relative to the absence of the coating layer.

82. The plasma nanoantenna according to any one of claims 79 to 81, wherein the coating layer comprises a plurality of oligonucleotides.

83. The plasma nanoantenna of claim 82, wherein each of the plurality of oligonucleotides comprises about 20 nucleotides to about 50 nucleotides.

84. The plasma nanoantenna of claim 83, wherein each of the plurality of oligonucleotides comprises about 20 nucleotides to about 30 nucleotides.

85. The plasma nanoantenna according to any one of claims 82 to 84, wherein each nucleotide of each of the plurality of oligonucleotides is selected from pyrimidine nucleotides.

86. The plasma nanoantenna according to any one of claims 61 to 85, wherein the nanoscale nucleic acid scaffold is substantially composed of double-stranded DNA, preferably composed of parallel interconnected double helical chains.

87. The plasma nanoantenna according to any one of claims 61 to 86, wherein the nanoscale nucleic acid scaffold is composed of a DNA origami structure.

88. The plasma nanoantenna of claim 87, wherein at least a portion of the DNA origami structure is assembled into a U-shaped or curved structure.

89. The plasma nanoantenna according to any one of claims 61 to 88, wherein the nanoscale nucleic acid scaffold comprises a first surface to which one of the two plasma nanoparticles is anchored, and a second of the two plasma nanoparticles is anchored to a second surface of the scaffold.

90. The plasma nanoantenna of claim 89, wherein the first surface of the scaffold and the second surface of the scaffold are located on the same side or adjacent side of the nanoscale nucleic acid scaffold.

91. The plasma nanoantenna of claim 89, wherein the first surface of the scaffold and the second surface of the scaffold are located on opposite sides of the nanoscale nucleic acid scaffold.

92. The plasma nanoantenna according to any one of claims 89 to 91, wherein the two plasma nanoparticles are respectively anchored to the first surface and the second surface of the scaffold via one or more nucleic acid adapters. Each nucleic acid linker is formed by an oligonucleotide coated on the surface of the plasma nanoparticles, and the oligonucleotide is capable of hybridizing with a polynucleotide forming part of or extending from the nanoscale nucleic acid scaffold. The sequences of the oligonucleotides and polynucleotides capable of hybridization are complementary or substantially complementary to each other.

93. The plasma nanoantenna according to any one of claims 61 to 92, wherein the nanoscale nucleic acid scaffold comprises one or more polynucleotides extending from the first side of the scaffold, each polynucleotide comprising a sequence complementary to the sequence of one or more oligonucleotides encapsulating one of the two plasma nanoparticles.

94. The plasma nanoantenna according to any one of claims 61 to 93, wherein the nanoscale nucleic acid scaffold comprises one or more polynucleotides extending from the second side of the scaffold, each polynucleotide comprising a sequence complementary to the sequence of one or more oligonucleotides encapsulating one of the two plasma nanoparticles.

95. The plasma nanoantenna of claim 93 or 94, wherein the one or more polynucleotides extending from the surface of the scaffold are independently selected from single-stranded DNA.

96. The plasma nanoantenna according to any one of claims 61 to 95, wherein the nucleic acid polymerase is bound to or fixed on the nanoscale nucleic acid scaffold via a nucleic acid adapter.

97. The plasma nanoantenna of claim 96, wherein the nucleic acid adapter is double-stranded and comprises: (i) a polynucleotide covalently or non-covalently bound to the nucleic acid polymerase via amino acids within the nucleic acid polymerase, and (ii) a polynucleotide forming part of or extending from the nanoscale nucleic acid scaffold, wherein the polynucleotides at (i) and (ii) comprise complementary or substantially complementary single-stranded DNA sequences capable of hybridizing with each other.

98. The plasma nanoantenna of claim 97, wherein the polynucleotide at (i) comprises a peptide tag, the peptide tag being bound to the nucleic acid polymerase via a heteropeptide bond formed between an amino acid within the nucleic acid polymerase and an amino acid within the peptide tag.

99. The plasma nanoantenna according to claim 97 or 98, wherein the nucleic acid adapter binds to the nucleic acid polymerase via an amino acid in the N-terminal domain of the nucleic acid polymerase.

100. The plasma nanoantenna according to any one of claims 61 to 99, wherein the nucleic acid polymerase is a DNA polymerase.

101. An array comprising a plurality of plasma nanoantennas according to any one of claims 61 to 100.

102. The array of claim 101, wherein the array comprises a solid substrate, and the plurality of said plasma nanoantennas are coupled on said solid substrate.

103. The array of claim 102, wherein each plasma nanoantenna is fixed to the solid substrate by its nucleic acid scaffold.

104. The array according to claim 102 or 103, wherein the solid substrate is glass or silicon dioxide, preferably in the form of a glass slide or chip.

105. The array according to any one of claims 101 to 104, wherein less than 30% of the plasma nanoantennas are aggregated into an aggregate of two or more plasma nanoantennas.

106. The array of claim 105, wherein less than 15% of the plasma nanoantennas are aggregated into an aggregate of two or more plasma nanoantennas.

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