Nanowire-assisted, single-molecule sequencing by synthesis

Abstract

The invention relates to a method, device, and substrate for single-molecule sequencing by synthesis utilizing one-dimensional (1D) light-guiding structures, such as nanowires, to guide fluorescent emissions for high-precision nucleotide detection. The method involves immobilizing target nucleic acids on the 1D structures, sequencing with fluorescently labeled nucleotides, and detecting emissions within the waveguiding field to ensure accuracy. Configurations such as reverse sequencing, securing nucleic acids along the structure, or immobilizing polymerase optimize detection fidelity. The substrate is designed for enhanced fluorescent detection, supporting applications in genomics, diagnostics, and biomedical research.

Description

[0001] Nanowire-assisted, single-molecule sequencing by synthesis

[0002] The present invention relates to sequencing by synthesis (SBS) and, more particularly, to methods and devices that use one-dimensional (1 D) light-guiding structures to improve detection of fluorescent emissions associated with nucleotide incorporation events.

[0003] Background of invention

[0004] The ability to accurately determine nucleic acid sequences is fundamental to advancements in genomics, diagnostics, and biomedical research. Sequencing by synthesis (SBS) is a widely adopted method for DNA sequencing due to its high throughput and sensitivity. In SBS, nucleotides are incorporated into a growing complementary strand, and their incorporation is detected by monitoring fluorescent signals emitted during each sequencing cycle. This approach has enabled significant progress in the study of genetic variation, disease mechanisms, and molecular biology. Despite its widespread use, SBS technologies face intrinsic limitations. One key challenge is the restricted length of accurately sequenced reads. Many current SBS platforms produce reads on the order of hundreds of base pairs with high accuracy, while sequencing longer regions is hindered by cumulative errors that arise during the iterative detection and nucleotide incorporation processes. In particular, limitations in fluorescence detection, such as background emission from unincorporated labeled nucleotides, optical scattering, and imperfect photon collection, can reduce signal-to-noise ratio and contribute to base-calling errors over repeated cycles. These errors result in a loss of sequencing fidelity over time, particularly when attempting to read extended genomic regions. As a result, short-read sequencing platforms are often inadequate for analyzing complex genomic features such as structural variants, repetitive regions, or rare mutations that require longer read lengths for accurate resolution.

[0005] The limited sequencing length in SBS presents challenges for many applications, including comprehensive genome assembly, analysis of complex genetic regions, and detection of subtle variations in clinically relevant genes. Additionally, the workflows required to maintain high accuracy in short-read sequencing systems often involve added complexity and cost, further limiting their scalability for certain applications.The need for sequencing platforms capable of achieving longer, highly accurate reads has become increasingly urgent. Improving sequencing fidelity, extending read lengths, and reducing errors associated with iterative processes are essential to addressing the demands of modern genomics and expanding the applicability of sequencing technologies to more complex and demanding use cases.

[0006] Summary

[0007] This present disclosure provides a method and device for sequencing by synthesis (SBS) that addresses key limitations of conventional sequencing platforms. By enabling precise single-molecule sequencing, the present disclosure can avoid amplification, thereby reducing errors and biases associated with cluster-based approaches. This can result in improved data fidelity, simplified workflows, and increased throughput.

[0008] The method improves signal detection efficiency and accuracy, allowing for reliable identification of nucleotide incorporation events with reduced phasing errors and minimized background noise. Furthermore, the system is designed to support parallel sequencing of multiple single molecules, enabling scalable and high-throughput applications.

[0009] By overcoming challenges such as limited read lengths and sequencing errors inherent in traditional SBS systems, the present disclosure provides a robust and efficient approach to nucleic acid sequencing, supporting advancements in genomics, diagnostics, and biomedical research.

[0010] The present disclosure therefore, in a first aspect, relates to a method of sequencing by synthesis, comprising:

[0011] • providing a one-dimensional (1 D) light-guiding structure having a subwavelength diameter, the 1 D light-guiding structure being configured to support one or more guided optical modes for propagating fluorescent emissions along its longitudinal axis;

[0012] • immobilizing a single-molecule target nucleic acid on or at a surface of said 1 D light-guiding structure;

[0013] • repeatedly introducing fluorescently labelled nucleotides under conditions allowing incorporation of each nucleotide into a growing strand complementary to the target nucleic acid;• detecting the fluorescent emissions from each incorporated nucleotide, wherein at least a portion of said emissions is coupled into the one or more guided optical modes and propagated along the longitudinal axis by the 1 D light-guiding structure; and

[0014] • determining the sequence of the target nucleic acid based on the detected emissions.

[0015] The present disclosure achieves technical advantages over conventional sequencing by synthesis methods. By employing a one-dimensional (1D) light-guiding structure with a sub-wavelength diameter, the present disclosure enables guidance of fluorescent emissions along the structure’s longitudinal axis. This can reduce signal loss and minimize background interference, facilitating detection of fluorescence from single nucleotide incorporation events.

[0016] The immobilization of single-molecule target nucleic acids on or at a surface of the 1 D structure can enable amplification-free sequencing in some embodiments, thereby reducing sequence biases and phasing errors commonly associated with cluster-based sequencing platforms. The detection of guided fluorescent emissions can provide improved signal-to-noise ratios, supporting reliable base calling and, in some embodiments, sequencing of longer DNA regions with improved accuracy.

[0017] Furthermore, the system’s scalability is enhanced by the potential to arrange multiple 1 D structures in dense arrays, enabling high-throughput sequencing of single molecules without compromising sensitivity or resolution.

[0018] In a further aspect, the present disclosure relates to a device for sequencing a nucleic acid by synthesis, comprising:

[0019] • a substrate bearing at least one one-dimensional (1 D) light-guiding structure configured to support one or more guided optical modes for propagating along its longitudinal axis;

[0020] • a flow cell in fluid communication with the substrate, enabling repeated introduction and removal of fluorescently labeled nucleotides and sequencing reagents;

[0021] wherein a single target nucleic acid molecule is immobilized on the 1 D lightguiding structure for sequencing.The device disclosed herein can be configured to facilitate the execution of the method described herein. By enabling single-molecule immobilization directly on the 1 D lightguiding structure, the device provides a platform for high-sensitivity sequencing workflows with minimal error rates and extended read lengths. The flow cell design allows for the introduction and removal of reagents in a controlled manner, making the device adaptable to various sequencing protocols.

[0022] In some embodiments, the device may be adapted to be used with, or incorporated into, existing sequencing platforms, offering a cost-effective solution to upgrade shortread systems to medium- and long-read capabilities. The modular nature of the device supports alternative configurations, such as varying the density or arrangement of the 1 D light-guiding structures, to optimize performance for specific sequencing applications.

[0023] The method and device disclosed herein provide a scalable, high-throughput approach to nucleic acid sequencing, offering significant improvements in accuracy and read length, with broad applicability in genomics, diagnostics, and biomedical research. In a further aspect, the present disclosure relates to a substrate for single-molecule sequencing by synthesis, comprising:

[0024] • a surface bearing at least one one-dimensional (1 D) light-guiding structure configured to support one or more guided optical modes for propagating fluorescent emissions along its longitudinal axis;

[0025] wherein each of said one or more 1 D light-guiding structures are functionalized to immobilize a single target nucleic acid molecule.

[0026] In a further aspect, the present disclosure relates to a system for sequencing by synthesis, comprising:

[0027] • a device as disclosed herein;

[0028] • a detection apparatus configured to capture fluorescent emissions guided by the one-dimensional (1 D) light-guiding structure; and

[0029] • a data processing unit operably coupled to the detection apparatus, the data processing unit being configured to analyze fluorescence data to generate sequence information of a target nucleic acid.

[0030] In a further aspect, the present disclosure relates to a method of manufacturing a device for sequencing by synthesis, comprising:• forming one or more one-dimensional (1 D) light-guiding structures on a substrate;

[0031] • functionalizing the 1 D light-guiding structures with a capture molecule capable of specifically binding to a complementary binding partner on a target nucleic acid; and

[0032] • optionally integrating the substrate into a flow cell configured to allow repeated introduction and removal of sequencing reagents.

[0033] In a further aspect, the present disclosure relates to a computer-implemented method for analyzing sequencing data, comprising:

[0034] • receiving fluorescence data generated from a device comprising one or more one-dimensional (1D) light-guiding structures;

[0035] • correlating fluorescence intensities with nucleotide incorporation events in a target nucleic acid molecule; and

[0036] • generating sequence information based on the correlated fluorescence intensities.

[0037] Brief description of the drawings

[0038] In the following embodiment and examples will be described in greater detail with reference to the accompanying drawings:

[0039] Fig. 1 shows a schematic view of the method of sequencing by synthesis, illustrating the steps from providing a 1 D light-guiding structure to determining the sequence of a target nucleic acid.

[0040] Fig. 2 illustrates the structure and arrangement of nanowires on a substrate, including views of the array, individual nanowires, and fluorescence imaging demonstrating their application in sequencing.

[0041] Fig. 3 depicts the waveguiding properties of a nanowire, showing light propagation along its longitudinal axis and highlighting the advantages for fluorescence detection. Fig. 4 provides a conceptual comparison of isotropic emission versus waveguided emission, emphasizing the improved light collection efficiency of nanowires.Fig. 5 shows a schematic of a flow cell platform with vertical nanowires, each functionalized to host a single target nucleic acid, and visualizes phasing errors during sequencing-by-synthesis workflows.

[0042] FIG. 6 schematically illustrates coupling of fluorescent emission from a fluorophore near a one-dimensional (1D) light-guiding structure into one or more guided optical modes.

[0043] FIG. 7 schematically illustrates an example excitation and / or detection arrangement for collecting guided emission from end regions of one or more 1 D light-guiding structures (73) on a substrate (74), using a light source (71) and collection optics (72).

[0044] Detailed description

[0045] In general, the present disclosure relates to sequencing-by-synthesis architectures that improve detection of single-molecule fluorescence signals. In some embodiments, fluorescent emission produced during nucleotide incorporation is coupled into one or more guided optical modes of a one-dimensional (1 D) light-guiding structure and transported along the structure toward a collection region. This guided-mode transport can increase the fraction of emitted photons that are collected by a detection apparatus and / or reduce collection of background fluorescence, thereby improving signal-to-noise and enabling more reliable base identification in sequencing-by-synthesis workflows. In contrast to approaches that rely primarily on localized confinement of an excitation or detection volume, the disclosed 1 D light-guiding structures can provide directional photon transport to a defined collection region, which may simplify optical collection requirements and improve detection efficiency.

[0046] The term "one-dimensional (1 D) light-guiding structure" refers to an elongated structure with a length greater than its transverse dimension(s), and configured to support propagation of optical energy along a longitudinal axis In some embodiments, a 1 D light-guiding structure comprises a dielectric or semiconductor body (or a hybrid / core-shell body) having a refractive index contrast relative to its surrounding medium sufficient to support one or more guided optical modes that confine optical energy at least in part to the structure and / or its evanescent field.

[0047] Examples include nanowires, nanorods, fibers, microwires, nanobelts, and tapered structures. In some embodiments, the 1 D light-guiding structure has a sub-wavelengthtransverse dimension (e.g., diameter), such that fluorescent emissions generated during sequencing can couple into the guided mode(s) and propagate along the structure toward a collection region; in some embodiments, photons are emitted from an end region (e.g., end facet) and collected by a detection apparatus.

[0048] For avoidance of doubt, the 1 D light-guiding structures disclosed herein are in contrast to “zero-mode waveguides” (ZMWs) and similar cut-off apertures formed in optically opaque films, which are typically operated in a regime where propagating modes are cut off and fluorescence detection is primarily localized near the aperture rather than being transported as guided emission along an elongated waveguiding element. In some embodiments of the present disclosure, single-molecule target nucleic acids are immobilized on or at a surface (e.g., a sidewall) of the 1D light-guiding structure such that nucleotide incorporation events occur within a region permitting coupling of fluorescent emission into one or more guided optical modes, enabling longitudinal photon transport and potentially improved collection efficiency and / or reduced background.

[0049] In some embodiments, fluorescent emission generated by a fluorophore (e.g., during a nucleotide incorporation event) is coupled into one or more guided optical modes of the 1 D light-guiding structure and transported along the longitudinal axis of the structure. Coupling may occur via near-field interaction between the fluorophore and the waveguiding mode of the structure, for example when the fluorophore is positioned sufficiently close to a surface of the 1 D light-guiding structure. The guided emission may propagate toward one or both ends of the 1 D light-guiding structure and couple out at an end region (e.g., an end facet) for collection by a detection apparatus.

[0050] The term “guided optical mode” refers to an optical field distribution supported by the 1 D light-guiding structure in which optical energy is confined at least in part to the structure and propagates along the longitudinal axis.

[0051] In some embodiments, the 1 D light-guiding structure extends from a substrate such that guided emission is collected from a distal end region, and / or from an opposite end region (e.g., through the substrate or from a rear side of the substrate).

[0052] The term “sub-wavelength diameter” (or sub-wavelength transverse dimension) refers to a diameter (or equivalent transverse dimension) that is less than the wavelength, in the surrounding medium, of fluorescent emission guided by the one-dimensional (1 D)light-guiding structure. In some embodiments, the relevant wavelength is a peak (or predominant) emission wavelength of a fluorescent label used in the sequencing reaction. For example, for fluorescent emission having a wavelength in the range of about 400 nm to about 800 nm, a sub-wavelength diameter may be in the range of about 10 nm to about 500 nm. In some embodiments, such sub-wavelength dimensions can increase coupling of fluorescent emission into guided mode(s), reduce isotropic scattering, and improve confinement and / or collection of emitted photons.

[0053] In some embodiments, a fluorophore associated with a nucleotide incorporation event is positioned within less than one-half of an emission wavelength, preferably less than one-tenth, from a surface of the 1 D light-guiding structure to promote near-field coupling

[0054] The term "sidewall functionalization" refers to the chemical or physical modification of the surface of the 1 D light-guiding structure to introduce bioreactive groups capable of selectively binding target nucleic acids or other biomolecules. Examples of functionalization techniques include silane-based chemistries or coating processes to immobilize specific binding molecules.

[0055] The term "capture molecule" refers to a molecule attached to the surface of the 1 D light-guiding structure that specifically binds to a complementary partner on the target nucleic acid. Examples of capture molecules include streptavidin, avidin, antibodies, aptamers, and nucleic acids.

[0056] In a first aspect, the present disclosure relates to a method of sequencing by synthesis. The method may comprise a step, such as a first step, of providing a one-dimensional (1 D) light-guiding structure having a sub-wavelength diameter. The 1 D light-guiding structure may be configured to guide fluorescent emissions along its longitudinal axis, facilitating efficient signal propagation and reducing isotropic scattering. It can be provided as part of a substrate or integrated into a flow cell. In some embodiments, the 1 D structure may be arranged in an array on the substrate to facilitate parallel sequencing of multiple single molecules. When included in a flow cell, the structure can be positioned to enable efficient reagent delivery and alignment with a detection apparatus. The structure may be implemented using nanowires composed of waveguiding materials, such as semiconductors or dielectrics, with a sub-wavelength diameter smaller than the emitted fluorescent light (e.g., 400-800 nm for visible light).In another step, such as a second step, the method may comprise immobilizing a single-molecule target nucleic acid. In this step, single-molecule target nucleic acids can be immobilized directly on the sidewall of the 1D light-guiding structure. Thus, single-molecule target nucleic acids may be immobilized or may be on or at a surface of said 1D light-guiding structure. This can be achieved through functionalization with capture molecules, such as streptavidin, avidin, or antibodies. Functionalization enables specific binding directly at the surface of the structure, ensuring that the target nucleic acids are securely anchored for sequencing. In some embodiments, each 1 D light-guiding structure may be limited to having a single target nucleic acid molecule attached to its surface. This configuration ensures that fluorescent emissions detected during sequencing correspond uniquely to the incorporation events of one molecule, avoiding signal overlap or ambiguity that could occur if multiple target sequences were present on the same structure. Reducing the number of target nucleic acid molecules per 1 D light-guiding structure, such as to a single molecule, can be achieved by optimizing the density of capture molecules on the sidewall to limit available binding sites. Alternatively, or additionally, the use of a dilute solution of target nucleic acids can reduce the likelihood of multiple molecules encountering and binding to the same structure. Blocking agents may also be introduced after the initial binding event to prevent any unoccupied sites from capturing additional nucleic acids.

[0057] In a further step, such as a third step, fluorescently labeled nucleotides, including reversible terminators, may be repeatedly introduced under conditions allowing singlenucleotide incorporation into a growing strand complementary to the target nucleic acid. The reversible terminators can block further extension temporarily, enabling controlled addition and detection of each nucleotide incorporation event. This repeated introduction of nucleotides ensures that sequential incorporation events are accurately detected.

[0058] In another step, such as a fourth step, fluorescent emissions generated during each nucleotide incorporation event can be detected, such as in real-time and / or by postcycle detection. The 1 D light-guiding structure may channel these emissions along its longitudinal axis, and the guided light can be emitted from an end region (e.g., end facet) of the structure. The emissions can be captured by a detection apparatus, such as a camera or photodiode array, ensuring efficient data acquisition and enabling the identification of incorporated nucleotides. The waveguiding properties of the 1 Dstructure may enhance signal-to-noise ratios, further improving the accuracy of fluorescence detection.

[0059] In some embodiments, coupling of fluorescent emission into the 1 D light-guiding structure produces directional emission (“beaming”) from an end region of the structure, thereby enabling preferential collection of guided emission relative to background fluorescence. In some embodiments, the detection optics are configured with a relatively low numerical aperture to preferentially collect beamed emission from the 1 D light-guiding structure while rejecting more isotropic background emission from unbound fluorophores or other sources.

[0060] In a further step, such as a fifth step, the sequence of the target nucleic acid may be determined based on the detected fluorescent emissions. A data processing unit can analyze the intensity, wavelength, and timing of the detected signals to generate base calls, correlating each signal to a specific nucleotide incorporation. By leveraging the waveguiding capabilities of the 1 D structure, the method may achieve high sequencing fidelity, minimize errors, and support long-read sequencing applications.

[0061] In one embodiment, the presently disclosed method may involve the use of fluorescently labeled nucleotides that function as reversible terminators. These nucleotides allow for the controlled incorporation of a single nucleotide into the growing strand during each cycle of sequencing. A fluorescent blocking group temporarily halts further strand extension after each incorporation, enabling precise detection of the corresponding fluorescent signal.

[0062] The term "fluorescently labeled nucleotides" refers to nucleotides chemically modified to include a fluorescent moiety. These moieties emit light upon excitation at a specific wavelength and are used to detect nucleotide incorporation events during sequencing by synthesis.

[0063] The term "fluorescent emissions" refers to light emitted from fluorescently labeled nucleotides upon excitation. These emissions are guided by the 1 D light-guiding structure to a detection apparatus for nucleotide identification.

[0064] The term "target nucleic acid" refers to a nucleic acid sequence, such as DNA or RNA, to be sequenced. In the present disclosure, the target nucleic acid is immobilized as a single molecule on the 1 D light-guiding structure.The term "base calling" refers to the process of interpreting detected fluorescent signals to determine the identity of the nucleotide incorporated during a sequencing cycle. Base calling typically involves computational analysis of fluorescence intensities and signal patterns.

[0065] The term "immobilizing" refers to the act of attaching or anchoring a molecule, e.g. a single target nucleic acid molecule to the sidewall of the 1 D light-guiding structure. This attachment is typically achieved through specific binding interactions enabled by the functionalization of the sidewall.

[0066] The blocking group can be removed after detection, allowing the sequencing process to proceed. Removal may be achieved through chemical, enzymatic, or photochemical reactions, depending on the specific reversible terminator used. This stepwise approach ensures controlled nucleotide addition, reducing errors caused by uncontrolled polymerase activity and improving base-calling accuracy.

[0067] By employing distinct fluorescent labels for each nucleotide base, the method allows for unambiguous identification of the incorporated nucleotide. This reversible terminator approach enhances sequencing precision while supporting high-throughput workflows and compatibility with various sequencing platforms.

[0068] In one embodiment, the presently disclosed method may be performed in a flow cell having an inlet and an outlet. The flow cell may be configured to facilitate the sequential delivery and removal of reagents, such as fluorescently labeled nucleotides, washing solutions, and blocking agents, ensuring precise control over the reaction environment during sequencing.

[0069] The inlet can introduce reagents in controlled volumes, while the outlet allows for the removal of spent or excess reagents, minimizing contamination and maintaining optimal reaction conditions for each cycle. This continuous flow system may enhance efficiency and ensure consistent reagent availability throughout the sequencing process.

[0070] By providing a controlled environment for reagent exchange, the flow cell may support high-throughput sequencing workflows and enable integration with detection systems for monitoring of fluorescent emissions. This design ensures accurate and efficient sequencing of single-molecule target nucleic acids.In one embodiment, the presently disclosed method may utilize a one-dimensional (1 D) light-guiding structure composed of a material suitable for waveguiding fluorescent emissions. Such materials may include semiconductors, dielectrics, or other waveguiding materials capable of effectively confining and propagating light along the longitudinal axis of the 1 D structure. The selection of material can depend on factors such as the wavelength of the fluorescent emissions, the required refractive index contrast, and the compatibility with functionalization techniques for immobilizing target nucleic acids. Semiconductor materials, such as silicon or gallium phosphide, may be particularly suitable due to their high refractive indices and well-established fabrication methods. Dielectric materials, such as titanium dioxide or silica, may also provide excellent optical transparency and low absorption for guiding light efficiently.

[0071] The use of materials suitable for waveguiding enables the 1 D structure to minimize signal loss, reduce isotropic scattering, and enhance the sensitivity of fluorescence detection. This improves the accuracy of single-molecule sequencing while supporting longer read lengths and reduced background noise. By employing materials optimized for waveguiding, the method may be adaptable to different fluorescence-based sequencing workflows and detection systems.

[0072] In one embodiment, the presently disclosed method may further specify that the semiconductor material used for the 1 D light-guiding structure is selected from the group consisting of silicon, germanium, silicon-germanium, gallium phosphide, gallium arsenide, gallium nitride, indium phosphide, indium arsenide, aluminum nitride, indium gallium arsenide, zinc oxide, zinc sulfide, zinc selenide, cadmium selenide, cadmium sulfide, titanium dioxide, tin oxide, lead sulfide, bismuth telluride, antimony telluride, perovskites, transition metal dichalcogenides, graphene, black phosphorus, and boron nitride. These materials are chosen for their high refractive indices, waveguiding efficiency, and compatibility with functionalization techniques.

[0073] For example, silicon is a well-established semiconductor material offering excellent waveguiding properties and ease of integration into microfluidic or flow cell systems. Materials such as gallium phosphide and indium phosphide are highly effective for guiding visible and near-infrared light, respectively, while zinc oxide and titanium dioxide provide strong optical transparency and high refractive index. Emerging materials, such as transition metal dichalcogenides and perovskites, offer novelproperties, including tunable optical behavior and enhanced fluorescence coupling, which may be advantageous in certain applications.

[0074] In one embodiment, the presently disclosed method may utilize a one-dimensional (1 D) light-guiding structure having a sub-wavelength diameter, such as of the emitted light. The diameter can, for example, be between about 10 nm and about 600 nm, such as 10 nm and 500 nm, or even 10 nm and 400 nm. A sub-wavelength diameter refers to a diameter smaller than the wavelength of the fluorescent emissions guided by the structure, which may typically range from about 400 nm to 800 nm for visible light. This size enables the confinement of light within the structure’s longitudinal axis, facilitating efficient waveguiding and minimizing isotropic scattering into the surrounding medium.

[0075] Diameters at the lower end of the range, such as 10-50 nm, may allow for strong confinement of light and high sensitivity to single-molecule fluorescence signals. Larger diameters, such as 300-600 nm, may be suitable for applications requiring reduced propagation losses or for guiding light at longer wavelengths, such as near-infrared emissions.

[0076] By maintaining a sub-wavelength diameter, the 1 D structure enhances the signal-to-noise ratio during sequencing by efficiently channeling emissions toward the detection apparatus. This geometry may also improve compatibility with high-resolution detection systems, such as cameras or photodiode arrays, by ensuring that the emitted light is well-focused and easily distinguishable from background fluorescence. The range of diameters further allows for optimization across different material choices and fabrication methods, supporting adaptability to various sequencing workflows.

[0077] In one embodiment, the presently disclosed method may involve a one-dimensional (1 D) light-guiding structure functionalized with a capture molecule capable of specifically binding to a complementary binding partner present on the target nucleic acid. Functionalization refers to the chemical and / or physical modification of the surface of the 1 D structure to introduce one or more active binding sites, enabling specific and stable attachment of target nucleic acids. This functionalization ensures precise immobilization of one or more single-molecule targets directly on the sidewall of the 1 D structure, which is critical for achieving accurate and high-resolution

[0078] sequencing.Capture molecules can include biomolecules such as streptavidin, avidin, neutravidin, antibodies, aptamers, or synthetic nucleic acid analogs like locked nucleic acids (LNAs) or peptide nucleic acids (PNA). These capture molecules are chosen for their ability to form strong and specific interactions with complementary binding partners, such as biotin, antigens, or complementary nucleic acid sequences. For example, a 1 D structure functionalized with streptavidin may specifically bind biotin-labeled target nucleic acids, forming a robust and reliable connection for sequencing workflows.

[0079] The use of specific capture molecules offers several technical advantages. First, it ensures that the fluorescent signals detected during sequencing are uniquely associated with the target nucleic acid, reducing background noise and preventing ambiguous signal interpretation. Second, functionalization provides versatility by allowing different binding chemistries to be tailored to the target nucleic acid’s characteristics, enabling broad applicability to various sequencing contexts. Lastly, the controlled immobilization of nucleic acids on the 1 D structure enhances the reproducibility of sequencing results, supporting high-throughput and scalable workflows. In some embodiments, the capture chemistry and / or spacer length is selected such that the nucleotide incorporation site is positioned within a near-field coupling region of the 1 D light-guiding structure, thereby promoting coupling of fluorescent emission into guided mode(s).

[0080] The capture molecule functionalization may be achieved through various techniques, such as silane-based surface chemistry, physical adsorption, or covalent attachment. The functionalized surface may also be treated to optimize the density of capture molecules, ensuring that only a single nucleic acid is immobilized per 1 D structure to avoid signal overlap or interference. By enabling precise and specific immobilization, this embodiment contributes to the method’s ability to deliver accurate single-molecule sequencing with minimal errors.

[0081] In one embodiment, the presently disclosed method may involve a one-dimensional (1 D) light-guiding structure that is doped to enhance waveguiding at the emission wavelength of the fluorescently labeled nucleotides. Doping refers to the introduction of impurities or modifications to the material of the 1 D structure to adjust its optical properties, such as refractive index or absorption coefficient. By optimizing these properties, the doped structure can improve light confinement and propagation efficiency along its longitudinal axis.For example, doping a semiconductor nanowire with elements such as phosphorus, boron, or arsenic may enhance its refractive index contrast, enabling more effective guidance of fluorescent emissions in the visible spectrum (e.g., 400-800 nm). In dielectric materials, such as titanium dioxide, doping can reduce scattering losses and improve transmission efficiency. By tailoring the doping concentration and type, the method ensures compatibility with the emission wavelength of the fluorescent labels, enhancing the signal-to-noise ratio and improving detection accuracy.

[0082] In one embodiment, the presently disclosed method may involve fluorescent emissions guided by the 1 D light-guiding structure being emitted from an end region (e.g., end facet) of the structure and detected by a detection apparatus positioned to capture the emitted light. The guided emissions may travel along the longitudinal axis of the structure and exit at its terminal edge, providing a focused and directed light signal for detection.

[0083] This arrangement allows for the efficient collection of light by a detection apparatus, such as a camera, photodiode array, or spectrometer, positioned in alignment with the end region. The directed nature of the emitted light reduces signal dispersion and background noise, ensuring high-resolution detection. Additionally, the use of the end region for light emission minimizes signal loss during propagation and simplifies alignment with the detection system. The presently disclosed method can allow for real-time detection and / or post-cycle detection.

[0084] In one embodiment, the presently disclosed method may involve a one-dimensional (1 D) light-guiding structure that is doped to enhance waveguiding at the emission wavelength of the fluorescently labeled nucleotides. Doping refers to the introduction of impurities or modifications to the material of the 1 D structure to adjust its optical properties, such as refractive index or absorption coefficient. By optimizing these properties, the doped structure can improve light confinement and propagation efficiency along its longitudinal axis.

[0085] For example, doping a semiconductor nanowire with elements such as phosphorus, boron, or arsenic may enhance its refractive index contrast, enabling more effective guidance of fluorescent emissions in the visible spectrum (e.g., 400-800 nm). In dielectric materials, such as titanium dioxide, doping can reduce scattering losses and improve transmission efficiency. By tailoring the doping concentration and type, themethod ensures compatibility with the emission wavelength of the fluorescent labels, enhancing the signal-to-noise ratio and improving detection accuracy.

[0086] In one embodiment, the presently disclosed method may involve fluorescent emissions guided by the 1 D light-guiding structure being emitted from an end region of the structure and detected by a detection apparatus positioned to capture the emitted light. The guided emissions may travel along the longitudinal axis of the structure and exit at its terminal edge, providing a focused and directed light signal for detection.

[0087] This arrangement allows for the efficient collection of light by a detection apparatus, such as a camera, photodiode array, or spectrometer, positioned in alignment with the end region. The directed nature of the emitted light reduces signal dispersion and background noise, ensuring high-resolution detection. Additionally, the use of the end region for light emission minimizes signal loss during propagation and simplifies alignment with the detection system. The presently disclosed method can allow for real-time detection and / or post-cycle detection.

[0088] In one embodiment, the presently disclosed method may yield nucleic acid reads with lengths of at least 200 base pairs, such as at least 400 base pairs. The ability to obtain longer reads is a significant advantage over conventional sequencing by synthesis methods, which are often limited by phasing errors and signal overlap.

[0089] Longer read lengths provide more comprehensive sequence information, enabling accurate detection of structural variations, repetitive regions, and rare mutations. This capability is particularly beneficial for applications such as de novo sequencing, haplotype phasing, and variant analysis. By leveraging the precise waveguiding and single-molecule detection capabilities of the 1 D structure, the method ensures reliable sequencing performance over extended DNA regions, reducing error rates and enhancing data fidelity.

[0090] In one embodiment, the presently disclosed method may involve detection being performed using a camera or photodiode array operably coupled to a data processing unit configured to generate base calls. The detection apparatus may capture the fluorescent emissions guided by the 1 D structure and convert the optical signals into electronic data.The data processing unit may analyze fluorescence intensity, wavelength, and timing to identify the incorporated nucleotide for each cycle. Algorithms can be employed to process the captured data, correct for potential errors, and generate base calls with high accuracy. This integration of detection hardware and computational analysis enables efficient, supporting high-throughput workflows while maintaining precision in base calling.

[0091] In one embodiment, the presently disclosed method may involve one or more onedimensional (1 D) light-guiding structures arranged on a substrate, wherein the arrangement can include configurations where the 1 D structures are perpendicular to a plane of the substrate. The substrate may feature a planar surface onto which the one or more 1 D structures are positioned, providing a stable and functional base for the sequencing process.

[0092] The arrangement of the 1 D structures on the substrate may be implemented as an ordered array, where the positions and orientations of the structures are systematically controlled, or as a disordered array, where the structures are stochastically distributed across the substrate surface. This flexibility allows for adaptation to different fabrication techniques and sequencing applications, ensuring broad applicability.

[0093] When the 1 D structures are oriented perpendicular to the substrate plane, the axial length of the structures can efficiently guide fluorescent emissions along their longitudinal axis toward a detection apparatus. This perpendicular orientation minimizes signal dispersion and ensures optimal coupling of fluorescent emissions into the waveguiding mode. In alternative embodiments, the 1 D structures may be arranged at different angles relative to the substrate plane, depending on the optical and functional requirements of the sequencing setup.

[0094] The substrate itself may be composed of materials such as glass, silicon, or polymers, chosen for their mechanical stability, optical transparency, and compatibility with fabrication processes. Surface treatments or coatings can further enhance the adhesion of the 1 D structures or reduce unwanted background fluorescence. Whether the 1 D structures are positioned in an ordered or disordered fashion, the substrate configuration supports efficient waveguiding, scalability for high-throughput workflows, and reliable single-molecule detection.In one embodiment, the presently disclosed method may involve co-immobilizing at least one polymerase enzyme alongside the target nucleic acid on the 1 D light-guiding structure. Co-immobilization ensures that the polymerase enzyme is positioned in close proximity to the target nucleic acid, facilitating efficient and localized nucleotide incorporation. The polymerase may be covalently attached, adsorbed, or otherwise bound to the functionalized surface of the 1 D structure, which is also used to immobilize the target nucleic acid.

[0095] This configuration offers several technical advantages. First, co-immobilization minimizes diffusion distances for nucleotide substrates, enhancing reaction kinetics and overall sequencing efficiency. Second, it ensures that the polymerase remains localized, reducing potential loss or degradation in solution during repeated reagent additions. Furthermore, this setup may improve signal specificity by ensuring that fluorescence emissions are confined to the target site, reducing background noise. The co-immobilization process can be achieved through dual-functionalization of the 1 D structure, where one set of capture molecules binds to the target nucleic acid, and another set binds to the polymerase. For example, biotinylated polymerases may be specifically attached to streptavidin-functionalized surfaces, or the polymerase may be linked via chemical crosslinkers. This approach ensures robust and precise attachment, enabling high-fidelity single-molecule sequencing.

[0096] In one embodiment, the presently disclosed method may employ four fluorescently labeled nucleotides, each corresponding to one of the four DNA bases — adenine, cytosine, guanine, and thymine. Each nucleotide may be labeled with a distinct fluorophore emitting light at a unique wavelength upon excitation. For example, adenine may emit in the blue region, cytosine in green, guanine in red, and thymine in the near-infrared, enabling distinct and separate detection of each base.

[0097] The use of four separately detectable fluorophores ensures that nucleotide incorporation events are accurately identified without ambiguity. This capability facilitates high-throughput sequencing while minimizing spectral overlap and cross-talk, leading to improved accuracy in base calling. The fluorophores may be selected based on their brightness, photostability, and compatibility with the optical properties of the detection apparatus and 1 D light-guiding structure. Examples include dyes from the Cy3, Cy5, Alexa Fluor, or Atto families. The labeled nucleotides may also incorporatereversible terminators to allow single-nucleotide incorporations per cycle, further enhancing precision.

[0098] In one embodiment, the presently disclosed method may involve maintaining the flow cell at a temperature optimized for the activity and stability of the polymerase enzyme. Temperature control is critical for ensuring efficient nucleotide incorporation, as the enzymatic activity of the polymerase is highly dependent on maintaining a suitable thermal environment. For many commonly used polymerases, the optimal temperature range may lie between 20°C and 40°C, although this can vary depending on the specific enzyme used.

[0099] The method may further involve synchronizing reagent additions, such as fluorescently labeled nucleotides or buffer solutions, with temperature control cycles to ensure that each step of the sequencing process occurs under optimal conditions. For example, reagent addition may occur during a stable temperature phase, followed by a temperature shift for nucleotide incorporation or blocking group removal. This synchronization may enhance the efficiency and accuracy of the sequencing workflow by ensuring that all reactions proceed under conditions ideal for their respective biochemical processes.

[0100] Temperature control may be achieved using external heaters, thermoelectric coolers, or integrated microfluidic systems within the flow cell. Monitoring systems, such as temperature sensors, may provide feedback to maintain the desired thermal environment. The combination of precise temperature regulation and synchronized reagent delivery reduces the likelihood of enzyme denaturation, incomplete nucleotide incorporation, or other errors, contributing to high-fidelity sequencing.

[0101] In one embodiment, the presently disclosed method may involve a one-dimensional (1 D) light-guiding structure configured to reduce background fluorescence by efficiently guiding relevant emissions along its longitudinal axis while suppressing isotropic scattering into the surrounding medium. The ability to confine light within the waveguiding mode ensures that fluorescent signals originating from nucleotide incorporation events are effectively transmitted to the detection apparatus with minimal loss or dispersion.

[0102] Background fluorescence, which arises from isotropic scattering or emissions from unintended sources, can interfere with accurate signal detection and reduce the signal-to-noise ratio. The 1 D structure's geometry and material properties, such as a high refractive index or optimized doping, enable it to channel light efficiently, minimizing leakage or stray emissions into the surrounding environment. By suppressing unwanted scattering, the structure ensures that the captured signals predominantly represent relevant fluorescence emissions, enhancing sequencing accuracy and fidelity.

[0103] The suppression of isotropic scattering may be further enhanced by the structure’s subwavelength diameter, which confines the electromagnetic field to a localized area near the structure’s surface. This enhances the coupling of fluorescence from surface-bound fluorophores into the waveguiding mode, while emissions not aligned with the structure's axis are effectively filtered out. For example, nanowires or nanorods composed of materials like silicon or gallium phosphide are particularly suited to this application due to their strong light-confining properties.

[0104] By reducing background fluorescence and isolating relevant emissions, the method improves the accuracy of base calling and extends the effective read length of the sequencing process. This enhanced signal clarity is especially critical for singlemolecule sequencing, where even minor noise can significantly impact the quality of the data. The ability of the 1 D light-guiding structure to achieve these advantages contributes to the method’s overall sensitivity, reliability, and applicability to high-throughput workflows.

[0105] In one embodiment, the presently disclosed method may involve functionalizing the sidewall of the one-dimensional (1 D) light-guiding structure using a chemistry suitable for anchoring bioreactive groups capable of selectively binding target nucleic acids. Functionalization methods may include silane-based chemistries, which are well-suited for materials like silicon nanowires due to their ability to form robust and stable surfaces with high loading capacity. Silanization can be carried out using reagents such as aminopropyltriethoxysilane (APTES) to introduce amine groups or other functional groups, enabling subsequent attachment of bioreactive molecules such as antibodies, streptavidin, or nucleic acid analogs.

[0106] In addition to silane-based methods, covalent bonding approaches may also be employed to attach functional groups to the surface of the 1 D structure. These approaches can include the introduction of carboxylic acid, epoxy, or aldehyde groups, which can then be used to form stable linkages with bioreactive molecules. Suchcovalent modifications can enhance the chemical stability of the functionalized surface, particularly under the operational conditions of nucleic acid sequencing workflows. Another functionalization approach may involve electrostatic interactions, where oppositely charged molecules are used to non-covalently bind bioreactive groups to the 1 D structure. For instance, polycationic or polyanionic coatings can serve as intermediate layers to facilitate the adsorption of biomolecules. This technique may be advantageous for applications requiring reversible functionalization or rapid surface modification.

[0107] While the described functionalization methods are broadly applicable to various 1 D structures, they are particularly relevant to silicon nanowires due to their high surface-to-volume ratio and compatibility with established surface modification techniques. For instance, functionalization with carboxylic acids or silane derivatives can enhance the nanowires’ optical and sensing properties, making them well-suited for guiding fluorescent emissions during sequencing. These methods also improve biocompatibility, enabling efficient and selective immobilization of target nucleic acids. Other materials, such as gallium phosphide or titanium dioxide, may also support functionalization through similar approaches. However, the available chemistries for these materials are relatively less developed compared to those for silicon-based nanowires, making silicon the preferred substrate for implementing the disclosed method. By selecting the appropriate functionalization chemistry, the method ensures precise, stable, and high-specificity binding of target nucleic acids, supporting the reliable operation of single-molecule sequencing workflows.

[0108] In one embodiment, the presently disclosed method may include processing fluorescence data obtained during nucleotide incorporation cycles using a base-calling algorithm. The algorithm may analyze the fluorescence intensities detected from each 1 D light-guiding structure to identify nucleotide incorporation events while assessing signal accuracy. By comparing intensity data, the algorithm can detect inconsistencies or deviations indicative of sequencing errors, such as misidentifications, signal overlap, or noise artifacts.

[0109] The base-calling algorithm can account for variables such as fluorescence intensity thresholds, timing discrepancies, and spectral overlap between fluorophores. It may incorporate error-correction methods, such as alignment to known sequences orstatistical models, to improve sequencing fidelity. For example, fluorescence intensities from multiple cycles may be compared to verify consistency in base calling, reducing the likelihood of misassignments.

[0110] By enabling real-time or post-processing error detection, the algorithm enhances the reliability of the sequencing results. This capability is particularly advantageous for single-molecule sequencing workflows, where signal variability may arise from minor inconsistencies in fluorophore labeling, incorporation efficiency, or photobleaching effects. The integration of error-detection mechanisms ensures that the sequencing output is accurate and robust, supporting downstream applications such as variant analysis.

[0111] In one embodiment, the presently disclosed method may include the storage of sequencing data in a computer-readable medium for subsequent analysis. The data may include information on base calls, fluorescence intensities, and timing from each incorporation cycle. Storing the data in a digital format allows for its retrieval, manipulation, and integration into various bioinformatics pipelines.

[0112] The stored sequence data can be compared to a reference genome or nucleic acid sequence to identify genetic variations, such as single nucleotide polymorphisms (SNPs), insertions, deletions, or structural variants. This comparison may involve alignment algorithms that match the obtained sequence data to the reference, highlighting differences indicative of genetic variants. By leveraging such comparisons, the method enables the identification of clinically or biologically significant mutations, facilitating research in genomics, diagnostics, and personalized medicine.

[0113] The computer-readable medium may include storage devices such as hard drives, solid-state drives, cloud-based storage systems, or other digital storage platforms. The ability to archive and analyze sequence data provides flexibility for large-scale genomic studies, multi-sample comparisons, and data sharing among research teams.

[0114] In one embodiment, the presently disclosed method may involve integrating sequencing results with protein expression data to enable a comprehensive analysis of genomic and proteomic interactions. Sequencing results, such as variant calls, insertion / deletion events, or structural rearrangements, may be aligned with datasets describing protein abundance, modifications, or interaction networks. This integration allows researchers to uncover correlations between genetic mutations and proteomicchanges, providing insights into the molecular mechanisms underlying various biological processes or diseases.

[0115] Computational frameworks may be employed to facilitate the integration of these datasets. Such frameworks may include tools for data normalization, alignment to reference genomic and proteomic databases, and statistical correlation analysis. For example, a mutation identified in a tumor suppressor gene via sequencing may be correlated with reduced expression or altered modification (e.g., phosphorylation) of its corresponding protein, suggesting a functional impact of the variant. These correlations may also extend to protein interaction networks, where mutations affect not only a single protein but entire pathways or complexes.

[0116] The ability to integrate sequencing and protein expression data provides significant advantages for proteogenomics studies, enabling researchers to connect genotype to phenotype and identify molecular drivers of disease. This approach supports applications in diagnostics, therapeutic target identification, and basic research into systems biology.

[0117] In one embodiment, the integration of sequencing results with protein expression data may be implemented using a computational pipeline designed to analyze complex, multi-omic datasets. This pipeline may align sequencing data to reference genomes or transcriptomes to identify genetic variations, including single nucleotide polymorphisms (SNPs), insertions, deletions, or larger structural variants. The aligned data provides a comprehensive map of genomic alterations, which can be linked to proteomic outcomes. Protein expression levels may be quantified using high-throughput proteomics techniques, such as mass spectrometry, fluorescence-based detection, or antibody arrays, assessing total protein abundance, post-translational modifications, or interaction patterns within protein complexes. Data integration may involve statistical methods or machine learning models to correlate genomic and proteomic datasets. For example, supervised learning models can identify patterns linking specific mutations to changes in protein expression levels, while unsupervised approaches may reveal novel relationships without prior assumptions. Techniques such as clustering or network analysis may identify broader impacts on pathways or interaction networks. This pipeline ensures relationships between genomic variations and proteomic profiles are accurately identified and interpreted, while computational models may detect complex, nonlinear relationships that conventional statistical methods could miss.By employing these techniques, the method enables the identification of genotypephenotype relationships, offering insights into the molecular basis of traits, diseases, and cellular responses. This integrated approach is particularly valuable for personalized medicine applications, where understanding the combined effects of genomic and proteomic changes can guide tailored therapeutic interventions.

[0118] In one embodiment, the presently disclosed method utilizes a 1 D light-guiding structure designed with a diameter optimized for waveguiding multiple wavelengths of fluorescent emissions. This approach enables efficient detection of distinct fluorophores corresponding to the four nucleotide bases used in sequencing by synthesis. The 1 D light-guiding structure, such as a nanowire, may be configured to capture light across a broad range of wavelengths, typically spanning the visible spectrum (e.g., approximately 400 nm to 800 nm). This broad-spectrum waveguiding capability ensures that emissions from all four nucleotide-specific fluorophores are efficiently guided and detected.

[0119] In some implementations, the diameter of the nanowire can be tailored to balance waveguiding efficiency across these wavelengths, optimizing the signal-to-noise ratio for each fluorophore. For example, nanowires with diameters in the range of 100 nm to 300 nm may be used to provide uniform waveguiding for all four fluorophores. This configuration reduces bias in fluorescence detection due to wavelength-dependent differences in coupling efficiency.

[0120] This multi-color waveguiding capability supports high-throughput sequencing by ensuring that emissions from all nucleotide incorporations are reliably detected, thereby enhancing the accuracy of base calling. It may also allow for flexibility in selecting fluorophores with different emission wavelengths, enabling further optimization of sequencing workflows.

[0121] In one embodiment, the presently disclosed method incorporates a 1 D light-guiding structure with a reflective end surface. This reflective end, implemented as a metallic or dielectric coating, is designed to reflect photons emitted within the waveguiding region of the structure back toward the opposite end. By reflecting photons that would otherwise exit the nanowire at one end, the reflective surface effectively doubles the photon signal available for detection at the opposite end of the nanowire. The term “waveguiding region” refers to a portion of the 1 D light-guiding structure and its near-field environment in which fluorescent emission can couple into guided optical mode(s) and be transported longitudinally.

[0122] The reflective coating may comprise materials such as aluminum, silver, gold, or other metals with high reflectivity in the relevant wavelength range. Alternatively, dielectric mirror coatings, composed of multilayered dielectric materials, may be used to achieve high reflectivity while minimizing optical losses. The reflective end surface can be engineered to align with the optical properties of the nanowire, ensuring maximum efficiency in photon reflection and retention within the waveguiding path.

[0123] By enhancing photon capture, this configuration improves the sensitivity and efficiency of fluorescence detection during sequencing. The approach reduces photon loss, leading to brighter and more reliable signals, which are critical for accurate base calling in single-molecule sequencing. Furthermore, the use of a reflective end does not alter the overall design or workflow of the sequencing system, making it compatible with existing platforms and detection apparatuses.

[0124] In a further aspect, the present disclosure relates to a device for sequencing by synthesis. The device may comprise a substrate bearing at least one one-dimensional (1 D) light-guiding structure configured to guide fluorescent emissions along its longitudinal axis. Thus, in some example, the substrate comprises multiple 1 D lightguiding structures. This, one or more, 1 D light-guiding structure may include nanowires / nanorods, or similar elongated structures with dimensions suitable for confining and propagating fluorescent emissions efficiently. The substrate may provide a stable support for the 1 D structures, ensuring their alignment and functionality during sequencing operations.

[0125] The device may additionally or alternatively comprise a flow cell in fluid communication with the substrate, enabling the repeated introduction and removal of fluorescently labeled nucleotides and sequencing reagents. The flow cell may include microfluidic channels, inlets, and outlets designed to facilitate the controlled delivery of reagents to the substrate. This configuration supports the iterative sequencing by synthesis process, allowing for precise reagent exchange and minimal dead volume. The flow cell design may further allow compatibility with standard sequencing workflows and detection apparatus.In one embodiment, the 1 D light-guiding structure may be functionalized with a capture molecule capable of specifically binding to a complementary binding partner on the target nucleic acid molecule. Functionalization enables selective attachment of the nucleic acid to the structure, facilitating precise single-molecule sequencing. Capture molecules may include proteins such as streptavidin, avidin, or neutravidin, which exhibit high affinity for biotin, as well as antibodies, aptamers, or nucleic acid analogs such as locked nucleic acids (LNA). Functionalization may be achieved through chemical modification of the 1 D light-guiding structure’s surface, such as silanization or polymer coatings, creating reactive sites for binding the capture molecules. In some embodiments, the capture molecule is immobilized via a coating, linker, hydrogel, or polymer layer disposed on the 1 D light-guiding structure. This selective attachment ensures that each fluorescent signal during sequencing corresponds to a specific nucleic acid molecule, reducing noise and signal overlap, thereby enhancing sequencing accuracy. In some embodiments, the functionalization is provided on a sidewall of the 1 D light-guiding structure such that the target nucleic acid is immobilized on or at the sidewall (e.g., directly or via one or more intermediate layers).

[0126] Functionalization methods may be tailored to suit the material properties of the 1 D light-guiding structure. For instance, silicon-based nanowires may be functionalized with silane-based chemistries, which provide robust attachment of bioreactive groups while maintaining waveguiding properties. In some embodiments, functionalization may also include crosslinking agents or spacer molecules to optimize binding efficiency and reduce steric hindrance during sequencing reactions.

[0127] In one embodiment, the target nucleic acid molecule may be functionalized with a complementary binding partner to enable specific binding to the 1 D light-guiding structure. Complementary binding partners may include biotin, antigens, or complementary nucleic acid sequences or other affinity partners (e.g., ligand-receptor pairs). For example, a target DNA strand may be biotinylated at its terminal region, allowing it to bind to streptavidin or avidin functionalized on the structure’s surface. Similarly, target nucleic acids may include sequences complementary to nucleic acid analogs, such as LNA or peptide nucleic acids (PNA), which can be immobilized on the structure.

[0128] This approach ensures a highly specific interaction between the target nucleic acid and the capture molecule on the structure, minimizing non-specific binding and maximizingthe efficiency of single-molecule immobilization. The use of complementary sequences also allows for precise orientation of the target molecule, facilitating efficient nucleotide incorporation and fluorescence detection during sequencing.

[0129] In one embodiment, the 1 D light-guiding structure may comprise a nanowire or similar elongated structure, such as a nanorod, with a sub-wavelength diameter between about 10 nm and 500 nm. The sub-wavelength diameter allows the structure to effectively confine and guide fluorescent emissions along its longitudinal axis. The length of the structure may range from about 1 pm to 50 pm, providing sufficient surface area for functionalization and optimal waveguiding efficiency.

[0130] Materials suitable for the 1 D light-guiding structure include semiconductors, such as silicon or gallium phosphide, or dielectric materials, such as titanium dioxide, which exhibit favorable optical properties for waveguiding. The structure’s geometry and composition may be optimized to enhance fluorescence signal propagation while reducing background scattering, ensuring high signal-to-noise ratios during sequencing.

[0131] In one embodiment, a plurality of 1 D light-guiding structures may be arranged on the substrate. These structures may be positioned either in an ordered array or in a disordered configuration, depending on the desired application. Ordered arrays may provide precise alignment for high-throughput sequencing and optimized signal detection, while disordered configurations may offer cost-effective manufacturing and flexibility in substrate design.

[0132] The arrangement of the 1 D structures may also consider their axial orientation relative to the substrate plane. For example, the structures may be positioned perpendicular to the plane to facilitate efficient collection of fluorescent emissions and minimize optical crosstalk. The spacing between adjacent structures may be optimized to prevent signal overlap during sequencing, ensuring reliable single-molecule detection.

[0133] In one embodiment, the device may include a flow cell comprising microfluidic channels configured for compatibility with standard sequencing platforms. The microfluidic channels may facilitate the controlled delivery and removal of reagents, such as fluorescently labeled nucleotides, polymerases, and washing buffers, to the substrate bearing the 1 D light-guiding structures.The flow cell design may incorporate features such as laminar flow dynamics, reagent mixing zones, and temperature control elements to optimize sequencing efficiency and minimize cross-contamination between reactions. Compatibility with standard sequencing platforms ensures seamless integration into existing workflows, enabling cost-effective upgrades to single-molecule sequencing capabilities without requiring extensive modifications to detection systems.

[0134] In one embodiment, the presently disclosed substrate comprises a configuration of the 1 D light-guiding structure, its functionalization, and the arrangement of the target nucleic acid molecule that is adapted to ensure that nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure during sequencing. The waveguiding region may correspond to an area near the surface of the 1 D lightguiding structure where the evanescent field supports efficient guidance of emitted fluorescent light. This adaptation can involve tailoring the functionalization density, the arrangement of capture molecules, and the spatial orientation of the target nucleic acid molecule relative to the 1 D light-guiding structure to maintain alignment with the waveguiding region. This configuration may enhance the precision of fluorescencebased sequencing by reducing signal loss and ensuring that detected emissions correspond to nucleotide incorporation events.

[0135] In one embodiment, the 1 D light-guiding structure may be functionalized to secure the target nucleic acid molecule along its sidewall, such that the nucleotide incorporation site remains within the waveguiding region of the 1 D light-guiding structure.

[0136] Functionalization can include the use of capture molecules, such as streptavidin or aptamers, or tethering agents specifically distributed along the sidewall. This arrangement ensures that the target nucleic acid molecule remains in close proximity to the light-guiding structure throughout the sequencing process, thereby reducing positional variability and maintaining consistent signal quality.

[0137] In one embodiment, the 1 D light-guiding structure may be configured to support reverse sequencing, wherein sequencing is initiated from a free end of the target nucleic acid molecule. By starting at the free end, the single-stranded region of the target nucleic acid remains within the waveguiding region of the 1 D light-guiding structure during sequencing. This configuration addresses challenges associated with the increasing rigidity and radius of gyration of double-stranded DNA formed during synthesis, which might otherwise extend beyond the waveguiding region. Reversesequencing may thus ensure optimal positioning of the fluorescent source relative to the light-guiding structure, enhancing the efficiency of signal detection.

[0138] In one embodiment, the substrate includes a DNA polymerase enzyme immobilized on the 1 D light-guiding structure in proximity to the target nucleic acid molecule. This proximity ensures that nucleotide incorporation events occur within the waveguiding region of the 1 D light-guiding structure, enabling efficient fluorescence detection. The immobilization of the polymerase may involve covalent attachment or other binding techniques that allow enzymatic activity to be retained while maintaining the spatial alignment needed for effective fluorescence guidance.

[0139] In one embodiment, the functionalization of the 1D light-guiding structure includes tethering agents or capture molecules distributed along its sidewall. These agents can serve to maintain the target nucleic acid molecule and associated sequencing components, such as the polymerase, within the waveguiding region during sequencing. The functionalization may involve chemical groups or coatings that provide specific binding sites for the target nucleic acid or related molecules. This arrangement not only enhances the stability of the sequencing setup but also ensures that nucleotide incorporation events consistently occur within the area of optimal fluorescence signal propagation.

[0140] In one embodiment, the presently disclosed method comprises adapting the configuration of the target nucleic acid molecule, the 1 D light-guiding structure, and associated sequencing components to ensure that nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure during sequencing. The waveguiding region may correspond to the area near the surface of the 1 D lightguiding structure where the evanescent field facilitates efficient guidance of emitted fluorescent light. By maintaining the nucleotide incorporation site within this region, the method may enhance the efficiency and reliability of fluorescence detection, ensuring accurate base calling and reducing background interference. This adaptation may involve optimizing the spatial alignment of the target nucleic acid molecule, the distribution of capture molecules, or the placement of sequencing components relative to the 1 D light-guiding structure.

[0141] In one embodiment, sequencing is initiated from a free end of the target nucleic acid molecule, such that the single-stranded region of the target nucleic acid remains within a waveguiding region of the 1 D light-guiding structure during sequencing. Thisapproach addresses challenges associated with the structural properties of doublestranded DNA, which has a larger radius of gyration and reduced flexibility compared to single-stranded DNA. By starting sequencing at the free end, the single-stranded DNA region being synthesized is consistently positioned within the waveguiding region, while the double-stranded portion remains outside this area. This configuration may improve the precision of fluorescence detection by ensuring that the fluorescent source remains in proximity to the 1 D light-guiding structure throughout sequencing.

[0142] In one embodiment, the target nucleic acid molecule is secured along the sidewall of the 1 D light-guiding structure using binding elements distributed along the structure, such that the nucleotide incorporation site remains within a waveguiding region of the 1 D light-guiding structure during sequencing. The binding elements may include capture molecules, such as streptavidin, aptamers, or other tethering agents, which are specifically configured to bind complementary functional groups on the target nucleic acid molecule. Securing the target nucleic acid molecule along the sidewall ensures that it maintains consistent alignment with the waveguiding region, thereby enhancing signal detection and reducing variability caused by positional shifts during sequencing.

[0143] In one embodiment, the DNA polymerase enzyme is immobilized on the 1D lightguiding structure in proximity to the target nucleic acid molecule, such that nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure. Immobilizing the polymerase on the structure may involve covalent bonding, adsorption, or other attachment methods that preserve enzymatic activity while ensuring spatial alignment. This configuration may reduce the likelihood of misalignment between the incorporation site and the waveguiding region, thereby improving the reliability of signal capture. The sequencing may be performed using fluorescently labeled reversible terminator nucleotides, which enable single-nucleotide additions and controlled fluorescence detection. By combining terminator chemistry with precise spatial control, this embodiment may facilitate high-fidelity sequencing with enhanced signal-to-noise ratios.

[0144] In one embodiment, the presently disclosed substrate for single-molecule sequencing by synthesis comprises one or more 1 D light-guiding structures, such as nanowires, designed to guide fluorescent emissions across multiple wavelengths corresponding to distinct fluorophores used in sequencing. The nanowires can have diameters optimizedfor efficient waveguiding of light emitted by fluorophores with emission spectra typically spanning from approximately 400 nm to 800 nm.

[0145] The nanowires may be fabricated with a uniform or gradient diameter to achieve balanced waveguiding efficiency for all fluorophores, ensuring accurate detection of all nucleotide-specific signals. For example, nanowires with diameters in the range of 100 nm to 300 nm can be configured to support waveguiding across the visible spectrum, minimizing bias in signal intensity due to wavelength-dependent differences. The nanowires may also be functionalized with capture molecules along their sidewalls to immobilize single target nucleic acid molecules, ensuring that fluorescent emissions occur within the waveguiding field.

[0146] This multi-wavelength waveguiding capability enhances the performance of sequencing workflows by supporting the detection of all fluorophores used in the method, thereby improving the accuracy and reliability of base calling. The substrate may also enable flexibility in selecting or modifying fluorophores to optimize sequencing performance without requiring changes to the underlying nanowire configuration.

[0147] In one embodiment, the presently disclosed substrate comprises one or more 1 D lightguiding structures with a reflective surface at one end of each structure. This reflective surface may include a metallic coating, such as aluminum, silver, or gold, or a dielectric mirror coating designed to reflect photons emitted within the waveguiding region of the nanowire. The reflected photons are redirected back toward the opposite end of the nanowire, where they can be captured by a detection apparatus.

[0148] The reflective surface can be engineered to align with the optical properties of the nanowire, such as its refractive index and diameter, ensuring maximum efficiency in photon reflection. This configuration effectively doubles the photon signal available for detection by preventing photon loss at the reflective end. The nanowires may also be functionalized with capture molecules to immobilize single target nucleic acid molecules and ensure that nucleotide incorporation events occur within the waveguiding field.

[0149] By incorporating reflective nanowire ends, the substrate provides enhanced signal intensity and improved sensitivity for fluorescence detection. This capability reduces signal loss and ensures more reliable detection of nucleotide incorporations, supporting high-accuracy sequencing workflows. The reflective nanowire design is compatible withexisting flow cell platforms and detection systems, making it a versatile enhancement to sequencing substrates.

[0150] In a further aspect, the present disclosure relates to a substrate for single-molecule sequencing by synthesis. The substrate may comprise a surface bearing at least one one-dimensional (1 D) light-guiding structure configured to guide fluorescent emissions along its longitudinal axis. Each 1 D light-guiding structure can be functionalized to immobilize a single target nucleic acid molecule, ensuring precise localization of the molecule on the structure. This configuration enables single-molecule sequencing by reducing background noise and preventing signal overlap from multiple molecules. By directly aligning with the inventive concept of using guided fluorescence to achieve accurate sequencing, this substrate provides a robust platform for scalable and high-throughput applications while maintaining single-molecule specificity.

[0151] In a further aspect, the present disclosure relates to a system for sequencing by synthesis. The system may comprise a device including one or more one-dimensional (1 D) light-guiding structures for guiding fluorescence, a detection apparatus configured to capture fluorescent emissions guided by the 1 D structures, and a data processing unit operably coupled to the detection apparatus. The data processing unit can analyze fluorescence data to generate sequence information of a target nucleic acid. By integrating these components, the system provides a comprehensive solution for sequencing by synthesis, enabling seamless detection, processing, and analysis of fluorescence signals. This approach ensures efficient signal interpretation, supporting applications requiring accurate base calling and long-read sequencing.

[0152] In a further aspect, the present disclosure relates to a method of manufacturing a device for sequencing by synthesis. The method may comprise forming one or more one-dimensional (1 D) light-guiding structures on a substrate, functionalizing these structures with a capture molecule capable of specifically binding to a complementary binding partner on a target nucleic acid, and optionally integrating the substrate into a flow cell configured for reagent delivery. This manufacturing process provides a scalable pathway for fabricating devices optimized for single-molecule sequencing. The functionalization of the 1 D structures ensures precise molecular attachment, while the integration into a flow cell facilitates compatibility with sequencing workflows. This method supports the development of cost-effective and high-performance devices for sequencing by synthesis.In a further aspect, the present disclosure relates to a computer-implemented method for analyzing sequencing data. The method may comprise receiving fluorescence data generated from a device with one or more one-dimensional (1 D) light-guiding structures, correlating fluorescence intensities with nucleotide incorporation events in a target nucleic acid molecule, and generating sequence information based on the correlated fluorescence intensities. This method leverages computational frameworks to interpret real-time fluorescence data accurately, enhancing base calling accuracy and minimizing sequencing errors. By aligning closely with the inventive concept, the computational method ensures that the signal-guiding and detection capabilities of the disclosed device are effectively utilized for high-fidelity sequence analysis.

[0153] The embodiments described herein are exemplary and are not intended to limit the scope of the present disclosure. Variations and modifications to the described embodiments may be made by those skilled in the art without departing from the scope of the invention as defined by the claims. For instance, while specific materials, dimensions, or functionalization methods are disclosed for the one-dimensional lightguiding structures, other suitable alternatives may be employed depending on the application or technological advancements.

[0154] Although the present disclosure is described in the context of sequencing by synthesis, the disclosed methods and devices may also be applicable to other fluorescencebased analytical techniques, such as single-molecule detection, molecular diagnostics, and biosensor development. The described technologies may find utility in genomics, proteomics, personalized medicine, and other fields requiring precise detection and analysis of nucleic acids or related biomolecules.

[0155] As used herein, terms such as 'may,' 'can,' 'optionally,' and 'in some embodiments' indicate that specific features are not required, but are permissive within the scope of the claims. Similarly, terms such as 'including,' 'comprising,' or 'such as' are intended to be non-limiting and indicate that additional elements may be present unless otherwise stated. References to 'one-dimensional light-guiding structures' should be understood to include, but not be limited to, nanowires, nanorods, and other elongated structures capable of waveguiding fluorescence emissions.

[0156] Detailed description of the drawingsIn the following sections, various embodiments of the present disclosure are further explained with references to the accompanying drawings. These drawings are provided to illustrate exemplary implementations of the present disclosure and should not be construed as limiting its scope. The features and configurations depicted in the drawings serve as examples and may be combined, modified, or adapted in accordance with the principles described herein to suit specific applications or use cases.

[0157] FIG. 1 is a flowchart illustrating the sequence of steps in the disclosed method of sequencing by synthesis. The process involves several sequential steps, beginning with providing a substrate (107) bearing one-dimensional (1 D) light-guiding structures (110) and continuing through detection of incorporation-associated fluorescence. Each step is labeled for clarity.

[0158] Step 101 illustrates the provision of one or more 1 D light-guiding structures (110) having a sub-wavelength transverse dimension. These structures, such as nanowires or similar elongated materials, are configured to support one or more guided optical modes for propagating fluorescent emissions (109) along their longitudinal axis, wherein at least a portion of the fluorescent emissions is coupled into the guided optical mode(s). The 1 D structures may be fabricated from semiconductor or dielectric materials and can be arranged on a substrate (107), optionally in ordered or disordered arrays.

[0159] Step 102 depicts the immobilization of single-molecule target nucleic acids (111 ) on or at a surface of the 1 D light-guiding structures (110), for example on or at sidewalls (113) via a functionalized surface and / or one or more intermediate layers (e.g., linkers or polymer coatings). The surface can be functionalized with capture molecules, such as streptavidin, avidin, antibodies, aptamers, and / or nucleic acid analogs, which specifically bind to complementary binding partners on the target nucleic acid. This immobilization can be configured such that each 1 D light-guiding structure (110) hosts a single target nucleic acid molecule (111).

[0160] Step 103 represents the repeated introduction of fluorescently labeled nucleotides (112) under conditions that allow their incorporation into a growing complementary strand (115). The labeled nucleotides may include reversible terminators, which temporarily block further synthesis after each incorporation, enabling controlled nucleotide addition and detection.Step 104 illustrates incorporation of nucleotides (112) into the complementary strand (115). Each incorporation event generates a fluorescent signal corresponding to the specific nucleotide added, forming the basis for sequence determination.

[0161] Step 105 describes detection of fluorescent emissions (109) from each incorporated nucleotide, wherein at least a portion of said emissions is coupled into the guided optical mode(s) and propagated along the longitudinal axis of the 1 D light-guiding structures (110). In some embodiments, the propagated emissions are emitted from one or more end regions (114) and captured by a detection apparatus.

[0162] FIG. 2A depicts a structured wafer substrate (107) designed to house vertically aligned nanowires (110) or similar elongated nanostructures. The substrate serves as a platform for sequencing workflows and is configured for compatibility with flow cell applications.

[0163] FIG. 2B presents an SEM image of an array of vertically aligned nanowires (110) on the substrate (107), showcasing their arrangement and uniformity.

[0164] FIG. 2C offers a magnified SEM view of individual nanowires (110), highlighting their dimensions and surface properties critical for waveguiding and functionalization.

[0165] FIG. 2D provides a tilted SEM view of the substrate (107) populated with vertically aligned nanowires (110), emphasizing their vertical orientation to enhance waveguiding efficiency.

[0166] FIG. 2E is a fluorescence image showing bright spots (108) corresponding to active fluorophores interacting with the nanostructures (110). These bright spots represent the fluorescently labeled nucleotides (112) incorporated during sequencing, demonstrating the nanostructures’ ability to guide emitted light (109) for precise signal collection. FIG. 3A illustrates the waveguiding properties of a nanowire (110). The central vertical structure represents the nanowire, with light (109) schematically depicting propagation within the structure.. This confinement ensures efficient guidance of photons along the nanowire’s axis, reducing signal loss and improving detection.

[0167] FIG. 3B provides a top-down view of the nanowire (110), schematically illustrating propagation of guided emission within the structure.. This perspective emphasizes how waveguiding properties maintain signal integrity and intensity during detection.FIG. 4A represents isotropic emission, where light (109) is emitted equally in all directions, leading to significant dispersion and signal loss.

[0168] FIG. 4B contrasts this with guided emission facilitated by the waveguiding properties of nanowires (110). Photons are directed into predefined paths along the structure’s axis, enhancing light collection efficiency and minimizing noise. In some embodiments, guided emission propagates toward one end or both ends of the structure.

[0169] FIG. 5 is a schematic representation of a substrate or flow-cell base (107) bearing an array of one-dimensional (1 D) light-guiding structures (110), such as vertically oriented nanowires. A single-molecule target nucleic acid (111 ) is immobilized on a surface of a respective 1 D light-guiding structure, for example on a sidewall (113). During sequencing-by-synthesis, nucleotides (112), including fluorescently labeled nucleotides, are incorporated to form a growing complementary strand (115).

[0170] Fluorescent emission generated at or near nucleotide incorporation events can couple into one or more guided optical modes of the 1 D light-guiding structure and propagate along the structure toward an end region / end facet (114) for collection by a detection apparatus (not shown). The inset illustrates a top view of an example arrangement of the 1D light-guiding structures.

[0171] FIG. 6 schematically illustrates an example mechanism by which fluorescent emission may couple into a one-dimensional (1 D) light-guiding structure. In the illustrated embodiment, one or more fluorophores are positioned on or near a surface of the 1 D light-guiding structure such that fluorescent emission generated by the fluorophores can couple into one or more guided optical modes of the structure, for example via near-field and / or evanescent-field coupling when the fluorophore is sufficiently close to the structure. The figure further schematically indicates refractive index regions (e.g., regions having refractive indices ni, n2, and an external medium n0) to illustrate refractive index contrast supporting guided mode propagation along the longitudinal axis of the 1 D light-guiding structure.

[0172] FIG. 7 schematically illustrates an example excitation and / or detection arrangement for use with one or more 1 D light-guiding structures (73) disposed on or extending from a substrate (74). A light source (71) may provide excitation light to excite fluorophores associated with nucleotide incorporation events occurring on or near the 1 D lightguiding structures (73). Fluorescent emission produced during such events may couple into one or more guided optical modes of a respective 1 D light-guiding structure (73)and propagate along its longitudinal axis toward an end region, from which the emission may be emitted and collected. Collection optics (72) (e.g., an objective or lens) are positioned to capture emission from end regions of the 1D light-guiding structures (73). In some embodiments, the collection geometry and / or numerical aperture of the optics (72) is selected to preferentially collect guided emission emitted from the end regions of the 1 D light-guiding structures (73) relative to more isotropic background emission from the surrounding medium.

[0173] FIG. 8A illustrates fluorescence imaging results obtained from a nucleotide incorporation assay performed on DNA hairpin constructs immobilized on nanowires. In this example, nanowire structures are functionalized with an extendable DNA hairpin (FIG. 8B) that serves as an intrinsic primer for polymerase-mediated nucleotide addition. Upon exposure of the nanowire-bound constructs to a sequencing reaction mixture containing a Cy5-labeled A nucleotide, a strong fluorescent signal is observed in the Cy5 detection channel (75). The pronounced emission indicates successful incorporation of the labeled nucleotide by the polymerase, confirming that the extendable hairpin is operative and capable of undergoing extension.

[0174] FIG. 9A illustrates fluorescence imaging results obtained from an analogous assay conducted using a hairpin construct bearing a 3'-terminal blocking group. In this configuration, the hairpin is rendered non-extendable by the presence of a

[0175] 3'-phosphate moiety (FIG. 9B), which prevents polymerase-mediated nucleotide incorporation. Imaging of nanowires treated with the same Cy5-labeled nucleotide mixture shows only weak fluorescent emission in the Cy5 channel (75). This minimal signal reflects the effectiveness of the 3'-blocking strategy in suppressing extension. The residual background fluorescence is attributed to nonspecific interactions of the dye-labeled nucleotide with the surface or DNA, rather than genuine incorporation.

[0176] Examples

[0177] The following examples are provided to illustrate certain embodiments of the present disclosure and are not intended to limit the scope of the claims. Unless otherwise indicated, the examples describe non-limiting implementations and variations and modifications are possible without departing from the present disclosure.

[0178] 1 : Illustrative 1D light-guiding substrate and SBS workflowIn one non-limiting example, a substrate comprises an array of one-dimensional (1 D) light-guiding structures extending from a surface, for example semiconductor or dielectric nanowires, which may be oriented vertically and / or laterally relative to the substrate. The 1 D light-guiding structures may have a transverse dimension in the range of about 10 nm to about 500 nm and a length in the range of about 1 pm to about 50 pm. A surface of the 1 D light-guiding structures is treated to enable immobilization of single target nucleic acid molecules, for example by attaching a capture molecule (e.g., streptavidin) and providing complementary binding partners on the target nucleic acids (e.g., biotin). The substrate may be integrated into a flow cell, and sequencing-by-synthesis may be performed by repeatedly introducing fluorescently labeled nucleotides, including reversible terminator nucleotides, under conditions allowing incorporation by a polymerase. Fluorescent emission produced during incorporation events may couple into one or more guided optical modes of a respective 1 D light-guiding structure and propagate along its longitudinal axis toward an end region (e.g., an end facet), where the emission is collected by a detection apparatus and processed to generate sequence information (e.g., base calls).

[0179] Example 2: Illustrative end-region collection, low-NA preferential detection, and optional reflector

[0180] In a further non-limiting example, guided emission is collected from an end region (e.g., a distal end facet) of a 1 D light-guiding structure using collection optics having a numerical aperture of about 0.7 or less, about 0.5 or less, or about 0.25 or less, thereby preferentially collecting emission propagated along the 1 D light-guiding structure relative to isotropic background emission from the surrounding medium. In some embodiments, excitation and / or collection optics are configured to preferentially collect emission emitted from the end region of the 1 D light-guiding structure. In some embodiments, a reflective surface (e.g., a metallic or dielectric coating) is provided at an end region of the 1 D light-guiding structure to reflect guided photons toward a detection end and increase collection efficiency.

[0181] Example 3: Nucleotide incorporation assay on nanowire-immobilized DNA hairpin constructs

[0182] Sequencing tests were conducted using a DNA hairpin construct configured to permit a nucleotide incorporation reaction, with the hairpin acting as an intrinsic primer. Hairpin oligonucleotides were prepared either in an extendable form (SeqHairpin 1 Cy5i54,Table 1) or in an extension-blocked form in which the 3' end was blocked by a 3' phosphate group (SeqHairpin 1 Cy5i54 Blocked, Table 1).

[0183] DNA constructs were immobilized on a nanowire SiO2surface using surface attachment chemistry. In this example, 2.5 mm x 2.5 mm silicon wafer pieces bearing nanowires were coated with a neutravidin layer by incubation for approximately 3 hours with a solution of 5 pg / mL neutravidin in PBS. The nanowire pieces were washed three times in TE buffer containing 0.1% Tween, incubated with 1 nM DNA hairpin oligonucleotides diluted in TE buffer for approximately 48 hours, and washed again three times in TE buffer containing 0.1% Tween.

[0184] The nanowire pieces were then incubated with 100 pL of a nucleotide incorporation mixture (MiSeq® Reagent Nano Kit v2 (300 cycles)) at approximately 60°C for approximately 1 hour. After incubation, the nanowire pieces were washed three times in TE buffer containing 0.1% Tween and imaged using an epifluorescence microscope in the Cy5 channel.

[0185] FIGS. 8A-8B show results obtained with the extendable hairpin construct. A strong fluorescent signal was observed in the Cy5 detection channel (75), consistent with incorporation of a Cy5-labeled nucleotide (e.g., an A base) by the polymerase. FIGS.

[0186] 9A-9B show results obtained with the 3'-blocked hairpin construct. Under otherwise similar conditions, substantially lower fluorescence was observed in the Cy5 channel (75), consistent with suppression of polymerase-mediated incorporation by the 3' blocking group. Any residual signal was attributed to background and / or non-specific association of dye-labeled nucleotides with the surface and / or nucleic acid constructs rather than incorporation.

[0187] This example is provided for illustration and is non-limiting; concentrations, times, temperatures, labels, and chemistries may be varied in other embodiments.

[0188] Table 1 : Oligonucleotide sequences

[0189]

[0190]

[0191] Items

[0192] 1. A method of sequencing by synthesis, comprising:

[0193] a) providing one or more one-dimensional (1 D) light-guiding structures having a sub-wavelength diameter, the 1 D light-guiding structures being configured to guide, or support one or more guided optical modes for propagating, fluorescent emissions along a longitudinal axis of the 1 D light-guiding structures;

[0194] b) immobilizing a single-molecule target nucleic acid on or at a sidewall of said 1 D light-guiding structure;

[0195] c) repeatedly introducing fluorescently labelled nucleotides under conditions allowing incorporation of each nucleotide into a growing strand complementary to the target nucleic acid;

[0196] d) detecting fluorescent emissions from each incorporated nucleotide, wherein at least a portion of said fluorescent emissions is coupled into the one or more guided optical modes and propagated along the longitudinal axis by the 1D light-guiding structure; and

[0197] e) determining the sequence of the target nucleic acid based on the detected emissions.

[0198] 2. The method according to item 1 , wherein the fluorescently labelled nucleotides are reversible terminators that allow a single nucleotide incorporation per cycle, followed by removal of a fluorescent blocking group.

[0199] 3. The method according to any one of the preceding items, wherein the method is performed in a flow cell having an inlet and an outlet for sequentially delivering and removing reagents.

[0200] 4. The method according to any one of the preceding items, wherein the 1 D lightguiding structure is a nanowire.

[0201] 5. The method according to any one of the preceding items, wherein the 1 D lightguiding structure is composed of a material suitable for waveguiding fluorescent emissions, such as a semiconductor material or a dielectric material.

[0202] 6. The method according to item 5, wherein the semiconductor material is selected from the group consisting of silicon, germanium, silicon-germanium, gallium phosphide, gallium arsenide, gallium nitride, indium phosphide, indiumarsenide, aluminum nitride, indium gallium arsenide, zinc oxide, zinc sulfide, zinc selenide, cadmium selenide, cadmium sulfide, titanium dioxide, tin oxide, lead sulfide, bismuth telluride, antimony telluride, perovskites, transition metal dichalcogenides, graphene, black phosphorus, and boron nitride.

[0203] 7. The method according to any one of the preceding items, wherein the 1 D lightguiding structure has a sub-wavelength diameter between 10 nm and 500 nm.

[0204] 8. The method according to any one of the preceding items, wherein the 1 D lightguiding structure is functionalized with a capture molecule capable of specifically binding to a complementary binding partner associated with the target nucleic acid.

[0205] 9. The method according to item 8, wherein the capture molecule is selected from streptavidin, avidin, neutravidin, or an antibody, aptamer or nucleic acid analog such as LNA and the complementary binding partner is selected from biotin, an antigen or the complementary nucleic acid sequence.

[0206] 10. The method according to any one of the preceding items, wherein the 1 D lightguiding structure is doped or otherwise modified to adjust refractive index and / or absorption at the emission wavelength.

[0207] 11. The method according to any one of the preceding items, wherein the fluorescent emissions guided by the 1 D light-guiding structure are emitted from an end facet of the structure and detected by a detection apparatus positioned to capture the emitted light.

[0208] 12. The method according to any one of the preceding items, wherein the fluorescent emission is coupled into the guided optical mode(s) via an evanescent field of the 1D light-guiding structure.

[0209] 13. The method according to any one of the preceding items, wherein the nucleic acid reads obtained have lengths of at least 200 base pairs, such as at least 400 base pairs.

[0210] 14. The method according to any one of the preceding items, wherein detection is performed using a camera or photodiode array operably coupled to a data processing unit configured to generate base calls.15. The method according to any one of the preceding items, wherein the one or more 1 D light-guiding structure are arranged on a substrate, such as wherein the one or more 1 D light-guiding structures are arranged perpendicular to a plane of the substrate.

[0211] 16. The method according to any one of the preceding items, wherein at least one polymerase enzyme is co-immobilized with the target nucleic acid on the 1 D light-guiding structure.

[0212] 17. The method according to any one of the preceding items, wherein the fluorescently labeled nucleotides are selected from a set of four distinctly labeled bases, each emitting at a different wavelength.

[0213] 18. The method according to any one of the preceding items, further comprising maintaining the temperature in the flow cell at an optimal range for the polymerase, and synchronizing reagent addition with temperature control cycles.

[0214] 19. The method according to any one of the preceding items, wherein detecting comprises collecting fluorescent emission from a first end region (e.g., a first end facet) and / or a second end region (e.g., a second end facet) of the 1 D lightguiding structure.

[0215] 20. The method according to any one of the preceding items, wherein the detecting comprises collecting guided emissions at an end facet while rejecting nonguided emissions from the surrounding medium.

[0216] 21. The method according to any one of the preceding items, wherein excitation and / or collection is performed with optics having a numerical aperture (NA) of at most about 0.7, at most about 0.5, or at most about 0.25.

[0217] 22. The method according to any one of the preceding items, wherein the sidewall functionalization is performed using a chemistry suitable for anchoring bioreactive groups capable of selectively binding target nucleic acids, such as silane-based chemistries, covalent bonding methods, polymer coatings, or electrostatic interactions.23. The method according to any one of the preceding items, wherein data from each incorporation cycle is processed by a base-calling algorithm that compares fluorescence intensities from each 1 D light-guiding structure to detect errors.

[0218] 24. The method according to any one of the preceding items, further comprising storing sequence data in a computer-readable medium and comparing the sequence to a reference for variant analysis.

[0219] 25. The method according to any one of the preceding items, wherein the sequencing results are integrated with protein expression data using computational frameworks to analyze relationships between genomic variations and proteomic profiles, such as correlations between genetic mutations and changes in protein abundance, modifications, or interaction networks.

[0220] 26. The method according to item 25, wherein the integration of sequencing results with protein expression data is performed using a pipeline that includes aligning genomic sequences to reference databases, quantifying protein expression levels, and employing statistical or machine learning models to identify genotype-phenotype relationships.

[0221] 27. The method according to any one of the preceding items, wherein the configuration of the target nucleic acid molecule, the 1 D light-guiding structure, and associated sequencing components is adapted to ensure that nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure during sequencing.

[0222] 28. The method according to item 27, wherein the nucleotide incorporation site is within an evanescent-field coupling distance of the 1 D light-guiding structure.

[0223] 29. The method according to item 27-28, wherein sequencing is initiated from a free end of the target nucleic acid molecule, such that the single-stranded region of the target nucleic acid remains within a waveguiding region of the 1 D lightguiding structure during sequencing.

[0224] 30. The method according to any one of items 27-29, wherein the target nucleic acid molecule is secured along the sidewall of the 1 D light-guiding structure using binding elements distributed along the structure, such that the nucleotideincorporation site remains within a waveguiding region of the 1 D light-guiding structure during sequencing.

[0225] The method according to any one of items 27-30, wherein the DNA polymerase enzyme is immobilized on the 1 D light-guiding structure in proximity to the target nucleic acid molecule, such that nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure, and wherein the sequencing is performed using fluorescently labeled reversible terminator nucleotides.

[0226] The method according to any one of the preceding items, wherein the 1 D lightguiding structure is designed with a diameter optimized to waveguide multiple wavelengths of fluorescent emissions corresponding to distinct fluorophores used for nucleotide incorporation events during sequencing.

[0227] The method according to item 31 , wherein the 1 D light-guiding structure is configured to efficiently capture and guide emissions from fluorophores emitting in the visible spectrum, such as wavelengths ranging from about 400 nm to about 800 nm.

[0228] The method according to any one of the preceding items, wherein the 1 D lightguiding structure comprises a reflective end surface configured to reflect photons emitted during nucleotide incorporation events, directing them toward the opposite end of the structure for enhanced detection efficiency.

[0229] The method according to item 34, wherein the reflective end surface is implemented as a metallic or a dielectric coating to optimize photon reflection and minimize photon loss, thereby improving the signal-to-noise ratio during sequencing.

[0230] A substrate for single-molecule sequencing by synthesis, comprising:

[0231] • a surface bearing at least one one-dimensional (1 D) light-guiding structure configured to support one or more guided optical modes for propagating fluorescent emissions along its longitudinal axis;

[0232] wherein each of the 1 D light-guiding structures are functionalized to immobilize a single target nucleic acid molecule.The substrate according to item 36, wherein the 1D light-guiding structure is functionalized with a capture molecule capable of specifically binding to a complementary binding partner on the target nucleic acid molecule, such as streptavidin, avidin, neutravidin, an antibody, aptamer, nucleic acid, or nucleic acid analog such as LNA or PNA.

[0233] The substrate according to any one of items 36-37, wherein the target nucleic acid molecule is functionalized with a complementary binding partner, such as biotin, an antigen, or a complementary nucleic acid sequence, to specifically bind to the functionalized 1 D light-guiding structure.

[0234] The substrate according to any one of items 36-38, wherein the 1 D light-guiding structure comprises a nanowire, or similar elongated structure with a subwavelength diameter between about 10 nm and 500 nm and optionally a length between about 1 pm and 50 pm.

[0235] The substrate according to any one of items 36-39, wherein a plurality of 1 D light-guiding structures are arranged on the substrate, either in an ordered array or in a disordered configuration.

[0236] The substrate according to any one of items 36-40, wherein the configuration of the 1 D light-guiding structure, a functionalization, and the arrangement of the target nucleic acid molecule are adapted to ensure that nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure during sequencing.

[0237] The substrate according to item 41, wherein the 1D light-guiding structure is functionalized to secure the target nucleic acid molecule along its sidewall, such that the nucleotide incorporation site remains within a waveguiding region of the 1D light-guiding structure.

[0238] The substrate according to any one of items 41 -42, wherein the 1 D light-guiding structure is configured to support reverse sequencing, such that sequencing is initiated from a free end of the target nucleic acid molecule, thereby ensuring that a single-stranded region of the target nucleic acid remains within a waveguiding region of the 1 D light-guiding structure during sequencing.

[0239] The substrate according to any one of items 41-43, wherein the 1 D lightguiding structure is adapted such that a DNA polymerase enzyme isimmobilized to the 1 D light-guiding structure in proximity to the target nucleic acid molecule, such that nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure.

[0240] 45. The substrate according to any one of items 41-44, wherein the functionalization includes tethering agents or capture molecules distributed along the sidewall of the 1 D light-guiding structure to maintain the target nucleic acid molecule and associated sequencing components within a waveguiding region.

[0241] 46. The substrate according to any one of items 36-45, wherein the 1 D light-guiding structure has a diameter optimized to support waveguiding across multiple wavelengths corresponding to distinct fluorescent emissions from sequencing reagents.

[0242] 47. The substrate according to item 46, wherein the diameter of the 1 D light-guiding structure is configured to guide light within a wavelength range of approximately 400 nm to 800 nm.

[0243] 48. The substrate according to item 46-47, wherein the 1 D light-guiding structure comprises nanowires with uniform or gradient diameters to balance waveguiding efficiency across all fluorophores used in sequencing.

[0244] 49. The substrate according to any one of items 36-48, wherein at least one end of the 1 D light-guiding structure comprises a reflective surface configured to reflect photons emitted within a waveguiding region of the structure back toward the opposite end.

[0245] 50. The substrate according to item 49, wherein the reflective surface comprises a metallic coating, such as aluminum, silver, or gold, or a dielectric mirror coating.

[0246] 51. The substrate according to any one of items 49-50, wherein the reflective surface is designed to maximize photon reflection efficiency based on the optical properties of the 1 D light-guiding structure.

[0247] 52. A device for sequencing by synthesis, comprising:

[0248] • The substrate according to any one of items 36-51 ;

[0249] • a flow cell in fluid communication with the substrate, enabling repeated introduction and removal of fluorescently labeled nucleotides and sequencing reagents;wherein a single target nucleic acid molecule is immobilized on the 1 D lightguiding structure for sequencing.

[0250] 53. The device according to item 52, wherein the flow cell comprises microfluidic channels configured for compatibility with standard sequencing platforms. 54. A system for sequencing by synthesis, comprising:

[0251] • a device according to any one of items 52-53;

[0252] • a detection apparatus configured to capture fluorescent emissions guided by the one-dimensional (1 D) light-guiding structure; and

[0253] • a data processing unit operably coupled to the detection apparatus, the data processing unit being configured to analyze fluorescence data to generate sequence information of a target nucleic acid.

[0254] 55. A method of manufacturing a device for sequencing by synthesis, comprising:

[0255] • forming one or more one-dimensional (1 D) light-guiding structures on a substrate, such as the substrate according to any one of items 34-49; • functionalizing the 1 D light-guiding structures with a capture molecule capable of specifically binding to a complementary binding partner on a target nucleic acid; and

[0256] • optionally integrating the substrate into a flow cell configured to allow repeated introduction and removal of sequencing reagents.

[0257] 56. A computer-implemented method for analyzing sequencing data, comprising:

[0258] • receiving fluorescence data generated from emissions propagated along the 1 D structure and collected at an end region;

[0259] • correlating fluorescence intensities with nucleotide incorporation events in a target nucleic acid molecule; and

[0260] • generating sequence information based on the correlated fluorescence intensities.

[0261] 57. The computer-implemented method according to item 56, wherein the fluorescence data is generated by the method according to any one of items 1- 35, and / or by the device according to any one of items 52-53.

Claims

49Claims1. A method of sequencing by synthesis, comprising:a) providing one or more one-dimensional (1 D) light-guiding structures having a sub-wavelength transverse dimension with respect to an emission wavelength, the 1 D light-guiding structures being configured to support one or more guided optical modes for propagating fluorescent emissions along a longitudinal axis of the 1 D light-guiding structures;b) immobilizing a single-molecule target nucleic acid on or at a surface of said 1 D light-guiding structure;c) repeatedly introducing fluorescently labelled nucleotides under conditions allowing incorporation of each nucleotide into a growing strand complementary to the target nucleic acid;d) detecting fluorescent emissions from each incorporated nucleotide, wherein at least a portion of said fluorescent emissions is coupled into the one or more guided optical modes and propagated along the longitudinal axis toward an end region by the 1 D light-guiding structure; ande) determining the sequence of the target nucleic acid based on the detected emissions.

2. The method of claim 1 , wherein the surface comprises a sidewall of the 1 D light-guiding structure.

3. The method according to any one of the preceding claims, wherein the singlemolecule target nucleic acid is immobilized on the surface via a functionalized surface and / or an intermediate layer.

4. The method according to any one of the preceding claims, wherein the fluorescently labelled nucleotides are reversible terminators that allow a single nucleotide incorporation per cycle, followed by deblocking, including removal of a blocking group and / or cleavage of a fluorescent label.

5. The method according to any one of the preceding claims, wherein the method is performed in a flow cell having an inlet and an outlet for sequentially delivering and removing reagents.

6. The method according to any one of the preceding claims, wherein the 1 D lightguiding structure is a nanowire.

507. The method according to any one of the preceding claims, wherein the 1 D lightguiding structure has a sub-wavelength transverse dimension between 10 nm and 500 nm.

8. The method according to any one of the preceding claims, wherein the 1 D lightguiding structure is functionalized with a capture molecule capable of specifically binding to a complementary binding partner associated with the target nucleic acid.

9. The method of claim 8, wherein the capture molecule is selected from streptavidin, avidin, neutravidin, an antibody, an aptamer, and / or a nucleic acid analog such as LNA or PNA, and the complementary binding partner is selected from biotin, an antigen, and / or a complementary nucleic acid sequence.

10. The method according to any one of the preceding claims, wherein the fluorescent emission is coupled into the guided optical mode(s) via an evanescent field of the 1D light-guiding structure.

11. The method of any of the preceding claims, wherein a fluorophore associated with a nucleotide incorporation event is positioned within less than one-half of a peak emission wavelength in the surrounding medium (A / 2), preferably within less than one-tenth of the peak emission wavelength (A / 10), from a surface of the 1D light-guiding structure.

12. The method according to any one of the preceding claims, wherein the fluorescent emissions guided by the 1 D light-guiding structure are detected at the end region of the structure.

13. The method according to any one of the preceding claims, wherein detecting comprises collecting fluorescent emission from a first end region and / or a second end region of the 1 D light-guiding structure.

14. The method of any preceding claim, wherein the detection apparatus is positioned to preferentially collect guided emission propagated along the 1D light-guiding structure relative to isotropic emission from the surrounding medium.5115. The method according to any one of the preceding claims, wherein excitation and / or collection is performed with optics having NA of 0.7 or less, 0.5 or less, or 0.25 or less.

16. The method according to any one of the preceding claims, wherein nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure during sequencing.

17. The method according to any one of the preceding claims, wherein at least one end of the 1 D light-guiding structure comprises a reflective surface configured to reflect photons emitted during nucleotide incorporation events toward the end region of the 1 D light-guiding structure.

18. The method according to claim 17, wherein the reflective end surface is implemented as a metallic or dielectric coating configured to reflect photons and reduce photon loss.

19. A substrate for single-molecule sequencing by synthesis, comprising a surface bearing at least one one-dimensional (1 D) light-guiding structure configured to support one or more guided optical modes for propagating fluorescent emissions along its longitudinal axis, wherein fluorescent emissions associated with a nucleotide incorporation event are couplable into the one or more guided optical modes and detectable at an end region of the 1 D light-guiding structure; wherein each 1 D light-guiding structure is configured to immobilize a single target nucleic acid molecule on or at a surface of the 1 D light-guiding structure.

20. The substrate according to claim 19, wherein the 1D light-guiding structure comprises a nanowire, or similar elongated structure with a sub-wavelength diameter between about 10 nm and 500 nm and optionally a length between about 1 pm and 50 pm.

21. The substrate according to any one of claims 19-20, wherein a plurality of 1 D light-guiding structures are arranged on the substrate, either in an ordered array or in a disordered configuration.

22. The substrate according to any one of claims 19-21 , wherein the configuration of the 1 D light-guiding structure, a functionalization, and the arrangement of the target nucleic acid molecule are configured such that nucleotide incorporation events occur within a waveguiding region of the 1 D light-guiding structure during sequencing.5223. The substrate according to any one of claims 19-22, wherein at least one end of the 1 D light-guiding structure comprises a reflective surface configured to reflect photons emitted within a waveguiding region of the structure back toward a detection end.

24. The substrate according to claim 23, wherein the reflective surface comprises a metallic coating, such as aluminum, silver, or gold, or a dielectric mirror coating.

25. A device for sequencing by synthesis, comprising:• the substrate according to any one of claims 19-24;• a flow cell in fluid communication with the substrate, enabling repeated introduction and removal of fluorescently labeled nucleotides and sequencing reagents;wherein the device is configured to detect fluorescent emissions propagated along the 1 D light-guiding structure toward an end region.

26. A system for sequencing by synthesis, comprising:• the device according to claim 25;• a detection apparatus positioned to capture fluorescent emissions emitted from an end region of the 1 D light-guiding structure; and• a data processing unit operably coupled to the detection apparatus, the data processing unit being configured to analyze fluorescence data to generate sequence information of a target nucleic acid.

27. A method of manufacturing a device for sequencing by synthesis, comprising:• forming one or more one-dimensional (1 D) light-guiding structures on a substrate, the 1 D light-guiding structures being configured to support one or more guided optical modes for propagating fluorescent emissions along a longitudinal axis;• rendering a surface of the 1 D light-guiding structures capable of immobilizing a target nucleic acid, including by functionalizing the surface with a capture molecule capable of specifically binding to a complementary binding partner on the target nucleic acid; and• optionally integrating the substrate into a flow cell configured to allow repeated introduction and removal of sequencing reagents.

Images