High-throughput analysis of single-molecule events

Abstract

The present disclosure relates to the analysis of single molecule events (40) observed by irradiating a sample (18) with excitation radiation (14) and detecting emission radiation (22) from the sample induced by the excitation radiation, wherein the paths of the excitation radiation from the radiation source (12) to the plurality of samples and the paths of the emission radiation from the plurality of samples to the detection means (26) do not overlap.

Description

[Technical Field]

[0001] explanation The present disclosure relates to the analysis of single molecule events observed by irradiating a sample with excitation radiation and detecting emission radiation from the sample induced by the excitation radiation, wherein the paths of the excitation radiation from the radiation source to the multiple samples and the paths of the emission radiation from the multiple samples to the detection means do not overlap. [Background technology]

[0002] background Sequencing the human genome or the genomes of other organisms and determining and comparing individual sequence variants requires the provision of sequencing methods that are firstly rapid and secondly can be used routinely and cost-effectively.

[0003] The high demand for cost-effective sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process and generate multiple sequences simultaneously. Examples of these sequencing technologies include massively parallel signature sequencing (Lynx Therapeutics), polony sequencing (Life Technologies), 454 pyrosequencing (Roche Diagnostics), Illumina sequencing (Solexa Inc), sequencing by ligation (Life Technologies), Ion Torrent semiconductor sequencing (Life Technologies), and DNA nanoball sequencing (Complete Genomics). These technologies enable rapid analysis of consensus sequences in a nucleic acid population. However, mutations present in minority sequences in the analyzed nucleic acid population, such as those present in a minority cellular genome, are often hidden by the large number of other sequences present in the population and therefore go undetected.

[0004] To address these issues, various forms of single-molecule analysis have been developed. Some of these processes fall within the field of fluorescence spectroscopy (FCS) and involve the detection and analysis of single molecules by fluorescence. Typically, in the case of DNA, DNA nucleic acid polymerases and / or nucleases and fluorescently labeled nucleic acid and / or nucleotide building blocks are used to individually determine the sequence of a single nucleic acid molecule based on the time-dependent changes in fluorescence as the nucleotide building blocks are incorporated into or cleaved from the nucleic acid molecule. Single-molecule sequencing processes and devices configured to perform such processes are described, for example, in WO2002 / 097406, WO2003 / 052137, WO2006 / 013110, WO2013 / 131888, WO2015 / 104245, WO2017 / 001407, and WO2018 / 104301.

[0005] In addition to DNA and / or RNA sequencing, there are several additional applications where high-yield single-molecule analysis is in high demand.

[0006] The disclosure US Pat. No. 7,259,847 B2, which is incorporated by reference, discloses a method for determining luminescent molecules by optical excitation in a confocal measurement volume, comprising the following steps: (a) providing a sample comprising luminescent molecules; (b) illuminating the sample with an optical excitation arrangement including a light source, a diffractive optical element for splitting the passing light into multiple focal points, and a focusing optical arrangement for focusing the passing multiple light beams into multiple confocal volume elements; and (c) Capturing emitted radiation from multiple confocal volume elements.

[0007] There is no disclosure of an embodiment in which the paths of the excitation radiation and the emission radiation do not overlap.

[0008] It was one of several objectives of the present disclosure to provide methods and apparatus for high-throughput analysis of single molecule events. Summary of the Invention

[0009] In a first aspect, the present disclosure relates to a method for analyzing single molecule events, said method comprising: (a) providing multiple samples in a field where single molecule events occur; wherein said plurality of samples are present on a support, wherein said support comprises an optically transparent substrate and a plurality of sample spots on a surface of the support, wherein the sample spots are spatially separated from one another, and wherein said samples are present on said sample spots; and providing a plurality of samples to a site where single molecule events occur, wherein a component of said single molecule event is immobilized on at least one sample spot, and wherein said immobilized component comprises a single biomolecule; (b) illuminating the plurality of samples with an optical excitation device including a radiation source, a diffractive optical element for splitting excitation radiation into a plurality of individual radiation beams, and an optical arrangement for directing the plurality of individual radiation beams toward the plurality of samples; (c) capturing radiation emitted from said plurality of samples with a detection means; and (d) analyzing the captured emission radiation, wherein a path of excitation radiation from said radiation source to said plurality of samples and a path of emission radiation from said plurality of samples to said detection means do not overlap, wherein said plurality of beams generated by an optical diffractive element pass to said plurality of samples through an optically transparent substrate of a support, wherein the plurality of samples are positioned on a surface of the support distal to a surface of the support through which excitation radiation enters; wherein each radiation beam of excitation radiation generates, by total internal reflection, each evanescent field on the surface of a support on which the plurality of samples are disposed; and The method wherein each individual evanescent field illuminates a group of sample spots.

[0010] In certain embodiments, the single molecule event comprises the sequencing of a single nucleic acid molecule.

[0011] A further aspect relates to an apparatus for analyzing single molecule events, said apparatus comprising: - a means for providing a plurality of samples configured for single molecule events, wherein said plurality of samples are present on a support, wherein said support comprises an optically transparent substrate and a plurality of sample spots on a surface of the support, wherein the sample spots are spatially separated from one another, and wherein said samples are present on said sample spots; and means for providing a plurality of samples configured for single molecule events, wherein a component of said single molecule event is immobilized on at least one sample spot, and wherein said immobilized component comprises a single biomolecule; an optical excitation device comprising a radiation source configured to emit excitation radiation, a diffractive optical element for splitting said excitation radiation into a plurality of individual radiation beams, and an optical arrangement for directing, e.g. focusing, said plurality of individual radiation beams onto said plurality of samples; - detection means configured to capture emitted radiation from said plurality of samples; and - analyzing means configured to analyze the captured emitted radiation; Including, wherein a path of excitation radiation from the radiation source to the plurality of samples and a path of emission radiation from the plurality of samples to the detection means do not overlap; wherein the apparatus is configured to pass the plurality of beams generated by the optical diffractive element through an optically transparent substrate of a support to the plurality of samples, wherein the plurality of samples are positioned on a surface of the support distal to a surface of the support through which the excitation radiation enters; wherein each radiation beam of excitation radiation generates, by total internal reflection, each evanescent field on the surface of a support on which the plurality of samples are disposed; and Here, each individual evanescent field illuminates a cluster of sample spots, The device.

[0012] In certain embodiments, the device is configured for the sequence analysis of a single nucleic acid molecule.

[0013] A further aspect relates to the use of the above method or the above device to provide high-throughput analysis of single molecule events. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows an embodiment of the present disclosure. A laser beam (12) is split into multiple individual beams, e.g., four individual beams (16), by a diffractive optical element (14). The individual beams (14) are directed toward a sample spot (18) disposed on a support (20), where the individual beams (14) generate a predetermined pattern of optical foci. Light (22) emitted from the foci at the sample spot (18) is directed toward a detector (26) by an optical arrangement (24). The difference between this embodiment and the prior art embodiment (previous slide) is that the optical path of the radiation source and the optical path of the emitted light are not the same throughout their paths and do not overlap. [Figure 2]FIG. 2 illustrates a further embodiment of the present disclosure in which detection is based on evanescent fields. In FIG. 2, a single evanescent field, e.g., one of a total of 10,000 evanescent fields, is depicted. An individual incident light beam (30), generated by passing excitation light through an optical diffraction element (not shown), passes through a gaseous medium (32) onto an optically transparent support (34), e.g., made of silicon dioxide. It enters the support (34) at a surface (34a) distal to a surface (34b) on which a sample spot (36) is located. Attached to the surface of the sample spot (36) are binding molecules (38) that mediate the immobilization of single biomolecules or single-molecule complexes (40), e.g., DNA or RNA polymerase, or complexes comprising DNA or RNA polymerase, a nucleic acid template, and a primer. The sample spot (36) is surrounded by a reaction space (42) containing a medium, such as an aqueous medium, and components necessary for single-molecule events, such as fluorescently labeled nucleotide building blocks. The incident light beam (30) is reflected from the surface (34b), generating a totally reflected light (44) and an evanescent field (46). The evanescent field (46) can encompass a single sample spot (36) or, as shown here, a group of sample spots (36), e.g., up to 100,000, and typically about 10,000, sample spots, and can have diameters ranging from 1 nm to 100 μm. Because the excitation light is split into multiple individual beams, multiple evanescent fields can be generated at the support surface (34b). Emission light from single-molecule events occurring on the sample spot (36) is passed to a detector (not shown). [Figure 3]3 illustrates a further embodiment of the present disclosure. A support (50) is provided having a sample spot (52) on its surface (not shown) with a single molecule immobilized thereon. The sample spot (52) is in contact with a single reaction space (54), which is a flow cell including an inlet (56) for the inflow of a medium containing fresh components for interaction with the single molecule, and an outlet (58) for the outflow of a medium containing spent components. Emitted light (not shown) from the sample spot (52) passes through the reaction space (54) to a detector (60). The detector includes pixels that are optically projected onto the sample spot (52) on the surface of the support (50). DETAILED DESCRIPTION OF THE INVENTION

[0015] Items in this specification 1. A method for analyzing single molecule events, the method comprising: (a) providing multiple samples in a field where single molecule events occur; (b) illuminating the plurality of samples with an optical excitation device including a radiation source, a diffractive optical element for splitting excitation radiation into a plurality of individual radiation beams, and an optical arrangement for directing the plurality of individual radiation beams toward the plurality of samples; (c) capturing radiation emitted from said plurality of samples with a detection means; and (d) analyzing the captured emitted radiation; Including, wherein the paths of excitation radiation from said radiation source to said plurality of samples and the paths of emission radiation from said plurality of samples to said detection means do not overlap. (Item 2) The method of item 1, wherein the path of the excitation radiation from the radiation source originates from a plane above the radiation path from the plurality of samples to the detection means. (Item 3) The method of item 1 or 2, wherein the plurality of samples are present on a support. (Item 4) The method of item 3, wherein the support comprises a substrate and a plurality of sample spots on the surface of the support, wherein the sample spots are spatially separated from one another, and wherein the sample is present on the sample spots. (Item 5) The method of item 4, wherein the substrate is optically transparent. (Item 6) The method of items 4 or 5, wherein a component of the single molecule event is immobilized on at least one of the sample spots. (Item 7) The method of Item 6, wherein the component to be immobilized comprises a single biological element, e.g., a single biomolecule. (Item 8) The method of any one of the preceding items, wherein the radiation source comprises at least one laser. (Item 9) The plurality of samples may be at least 10, at least 100, at least 1,000, or at least 10 4 , at least 10 5 , or at least 10 6 , and up to 10 8 , 10 9 , up to 10 10 , or up to 10 11 10. The method of any one of the preceding items, comprising an individual sample of (Item 10) The method of any one of the preceding items, wherein the individual radiation beams of excitation radiation are split into about 10 to about 1000, or about 50 to about 500, e.g., about 100, individual radiation beams. (Item 11) The method of any one of the preceding items, wherein individual radiation beams of excitation radiation are directed at an individual sample or a group comprising multiple individual samples. (Item 12) Each radiation beam of excitation radiation is about 10 to about 10 containing a plurality of individual samples. 6 , or from about 100 to about 10 5 The method of any one of the preceding items is directed to the group of (Item 13) A group can contain up to about 10 6 individual samples, and in particular from about 1,000 to about 10 6 , or about 5,000 to about 10 5 13. The method of item 11 or 12, comprising, for example, about 10,000 individual samples. (Item 14) The method of any one of the preceding items, wherein step (a) includes focusing the plurality of individual radiation beams onto the plurality of samples. (Item 15) The method of any one of the preceding items, wherein the multiple beams generated by the optical diffraction element are passed to the multiple samples through an optically transparent substrate of a support on which the multiple samples are placed. (Item 16) The method of item 15, wherein the plurality of samples are disposed on a surface of the support that is distal to the surface of the support through which the excitation radiation enters. (Item 17) The method of item 16, wherein each radiation beam of excitation radiation generates a separate evanescent field on the surface of the support on which the multiple samples are disposed. (Item 18) The method of item 17, wherein the individual evanescent fields have a diameter of about 1 nm to about 100 μm, or about 10 nm to about 10 μm. (Item 19) The individual evanescent fields are at most about 10 6 individual samples, and in particular from about 1,000 to about 10 6 19. The method of item 17 or 18, covering from about 5,000 to about 20,000, e.g., about 10,000 individual samples. (Item 20) The method of any one of Items 17 to 19, wherein the individual evanescent fields are generated via total internal reflection. (Item 21) The method of any one of items 17 to 20, wherein a plurality of individual evanescent fields are generated. (Item 22) The method of item 21, wherein the individual evanescent fields do not substantially overlap. (Item 23) The method of item 21, wherein the overlap between two adjacent fields is about 20% (by area) or less, or about 10% (by area) or less. 24. The method of any one of the preceding claims, wherein the diffractive optical element is an optical diffraction grating. (Item 25) The method of any one of the preceding items, wherein the detection means includes a detector matrix including a plurality of detection pixels. (Item 26) The method of item 25, wherein the detection pixels are optically projected onto sample spots on the surface of the support. 27. The method of any one of the preceding claims, wherein the detection means provides an optical focus on each individual sample spot. (Item 28) The method of any one of the preceding items, wherein at least one reaction space is provided around the plurality of samples, wherein the reaction space contains a medium and components for the single molecule event. (Item 29) The method of Item 28, wherein the reaction space is a flow cell. (Item 30) The method of Item 28 or 29, wherein a single reaction space is provided around the multiple samples. (Item 31) The method according to any one of Items 28 to 30, wherein the reaction space has a depth of about 10 nm to about 1,000 μm. (Item 32) The method according to any one of Items 28 to 31, wherein the path of the radiation passes through the reaction space. (Item 33) The method of any one of the preceding items, wherein the single molecule event comprises sequence analysis of a single nucleic acid molecule. 34. An apparatus for analyzing single molecule events, comprising: - means for providing a plurality of samples configured for single molecule events; - an optical excitation device including a radiation source configured to emit excitation radiation, a diffractive optical element for splitting the excitation radiation into a plurality of individual radiation beams, and an optical arrangement for directing the plurality of individual radiation beams towards the plurality of samples; - detection means configured to capture emitted radiation from said plurality of samples; and - analyzing means configured to analyze the captured emitted radiation; Including, wherein a path of excitation radiation from the radiation source to the plurality of samples and a path of emission radiation from the plurality of samples to the detection means do not overlap; The device. (Item 35) The apparatus of Item 34 configured to perform the method of any one of Items 1 to 33. (Item 36) The device of item 34 or 35 configured to perform single molecule nucleic acid sequence analysis. (Item 37) Use of the method of any one of items 1 to 33 or the device of any one of items 34 to 36 to provide high-throughput analysis of single molecule events. (Item 38) High-throughput analysis is about 10 6 from about 10 11 Use of item 37, including parallel analysis of individual samples. (Item 39) High-throughput analysis allows for approximately 10 6 from about 10 11 Use of item 38, including parallel analysis of individual sample spots. (Item 40) Use of Items 38 or 39, in which high-throughput analysis is performed in a single reaction space.

[0016] Detailed Description The present disclosure provides methods and devices for analyzing single molecule events, where the single molecule events are associated with the emission of electromagnetic radiation from a sample, e.g., a sample spot on a support containing components of the single molecule event. In certain embodiments, the single molecule event involves a reaction of a biological component associated with the emission of electromagnetic radiation having a characteristic.

[0017] The present disclosure addresses the need to provide improved high-throughput methods for the analysis of single molecule events observed by irradiating a sample with excitation radiation and simultaneously detecting emission radiation arising from the sample induced by said irradiation. The concepts described in this disclosure involve the parallel analysis of multiple groups of individual sample spots on a single support, each group containing up to 10 6 diffractive optical element that splits the excitation radiation beam can have, for example, up to 10 sample spots, where each group can be illuminated by a single evanescent field. 4 , up to 10 5 Thus, the total number of sample spots analyzed according to the present disclosure can be greater than about 10, particularly on a single support. 6 , up to about 10 11Additionally, the present disclosure provides a reaction space containing a liquid medium in contact with the sample solution, e.g., a sample spot, that requires a smaller volume allowing for greater flexibility in sample solution distribution.

[0018] In particular, the present disclosure provides an apparatus and process for irradiating a plurality of samples with excitation radiation produced by a radiation source, for example a radiation source including at least one laser, and split into a plurality of individual radiation beams by an optical diffractive element, wherein the plurality of individual radiation beams are directed at said plurality of samples, the samples comprising components capable of emitting emission radiation that is intercepted and analyzed by detection means.

[0019] According to the present disclosure, the paths of excitation radiation from the radiation source to the multiple samples and the paths of emission radiation from the multiple samples to the detection means do not overlap, which makes it possible to obtain a much larger surface area on which the sample spots are located and therefore greatly increases the number of samples that can be analyzed.

[0020] In some embodiments, the path of excitation radiation from the radiation source originates from above, i.e., from a plane spatially above the radiation paths from the plurality of samples to the detection means.

[0021] In some embodiments, the plurality of samples is at least 10, at least 100, at least 1,000, at least 10 4 , at least 10 5 , or at least 10 6 , and up to 10 8 , 10 9 , up to 10 10 , or up to 10 11 Contains individual samples of

[0022] The sample is typically present on a support. The support may include a substrate and a plurality of sample spots on the support surface that are spatially separated from one another. In certain embodiments, the support surface is formed by the substrate and the sample spots, and the sample is present on the sample spots. In certain embodiments, the substrate forms a continuous area in which the sample spots are distributed. Each sample spot on the support surface is surrounded by a substrate, which differs from the sample spots, for example, in terms of material and / or surface. Typically, the sample spots are configured to allow attachment of desired biomolecules, while the substrate is configured to inhibit and / or prevent attachment of biomolecules such as polypeptides.

[0023] On the sample spot, a component, e.g., a biological component, involved in the single molecule event is immobilized. In certain embodiments, the component comprises a biomolecule, particularly a single biomolecule. The term "single biomolecule" encompasses a single molecular entity, such as a polypeptide, or a complex consisting of multiple individual units, e.g., individual molecular entities, where the individual units together form a functional biological component.

[0024] In certain embodiments, the support is a substantially planar support, i.e., does not contain any peaks or valleys of about 1000 nm or more, or about 100 nm or more. In further embodiments, the support is a structured support, e.g., a surface area of ​​about 5×10 -24 liters to approximately 1 x 10 -15 The support may include well-like recesses having a volume of up to 1 liter, or pillars having a height of about 1 nm to 500 nm. In principle, the support may have any design, as long as a reaction space can be formed that allows the occurrence of a single molecule event on at least two of the sample spots on which a single biomolecule is immobilized.

[0025] In certain embodiments, the substrate is an optically transparent material, i.e., a material that is substantially transparent to electromagnetic radiation, e.g., radiation in the visible range and / or radiation in the near-infrared range. In certain embodiments, the substrate comprises a material having an absolute refractive index of at least 1.01, e.g., an absolute refractive index of about 1.5 to about 3 in the visible range, or about 1.5 to about 4 in the near-infrared range. In further embodiments, the substrate is an optically opaque material, e.g., a metal or a metalloid such as silicon.

[0026] In certain embodiments, the substrate comprises a non-conductive material. Specific examples are glass, quartz, plastic, a metal oxide-based material, e.g., a silicon dioxide-based material (such as glass, silica, or quartz), or a composite material comprising the above materials. In further embodiments, the substrate comprises a conductive material, e.g., an optically transparent material such as indium tin oxide.

[0027] Typically, the substrate has a thickness of about 10 μm to about 5 mm, particularly about 20 μm to about 2 mm.

[0028] The surface of the support includes a plurality of sample spots that are spatially separated from one another by the substrate surface. The sample spots are configured for attachment of biomolecules. In some embodiments, the support includes a plurality of sample spots, e.g., at least 10, at least 100, at least 1,000, at least 10 4 , at least 10 5 , or at least 10 6 , and up to 10 8 , 10 9 , up to 10 10 , or up to 10 11 Each sample contains individual sample spots.

[0029] In some embodiments, the sample spot comprises or consists of at least one conductive material, such as a single metal, or a combination of metals, such as an alloy or mixture of different metals. For example, metals that can be attached to sulfur-containing elements in the form of thiols or disulfides, or metals that can be attached to chelating elements such as polyhistidine tags, are suitable. In some embodiments, the metal has a positive electrochemical potential. Specific examples of suitable metals include, but are not limited to, Au, Cu, Ni, Pt, Pd, Rh, Ir, Os, Ru, and any combinations thereof comprising at least two of the foregoing metals.

[0030] In some embodiments, the sample spot comprises or consists of at least one metal oxide, including a single metal oxide or a combination of metal oxides. For example, metal oxides that can be attached to phosphorus-containing elements in the form of phosphonic acids or phosphonate esters, or metal oxides that can be attached to chelating elements such as polyhistidine tags, are suitable. Specific examples of suitable metal oxides include TiO2 and NiO.

[0031] In a further embodiment, the sample spot comprises or consists of at least one non-conductive material.

[0032] Sample spots can be prepared by deposition of metal vaporized on a substrate covered with a grid mask, which can be generated by electron beam lithography or equivalent techniques. The size of the openings in the grid mask can correspond to the size of the spots on the substrate surface. Alternatively, spots on the substrate can be prepared by site-specific deposition of nanoparticles, e.g., with a size of 2-10 nm, onto the substrate, particularly substrates with planar surfaces, by precision pipetting of the particles.

[0033] In certain embodiments, the sample spots have a size suitable for attachment of a single biomolecule, hi such embodiments, the sample spots have a diameter of about 1 nm to about 30 nm, particularly about 2 nm to about 20 nm.

[0034] In some embodiments, the sample spot is a discrete structure on the surface of the substrate. In some embodiments, the sample spot has a lower surface proximal to the substrate and an upper surface distal to the substrate, where the distance between the lower surface and the upper surface defines the height of the sample spot. In certain embodiments, the height is about 50 pm to about 500 nm, particularly about 100 pm to about 20 nm, and more particularly about 500 pm to about 10 nm, for example, about 2 nm.

[0035] The sample spots on the support can be configured for the attachment of biomolecules, for example, by covalent or non-covalent bonds. The biomolecules can be selected from polypeptides, nucleic acids, carbohydrates, and any combination thereof, for example, glycosylated polypeptides or ribonucleoproteins. In some embodiments, the biomolecule is a complex consisting of several individual units, for example, several polypeptide units, or several polypeptide and nucleic acid units.

[0036] In a specific embodiment, the biomolecule is a nucleic acid polymerizing enzyme, particularly a DNA polymerase or an RNA polymerase, or a nucleic acid-polymerizing molecule complex, particularly a DNA polymerizing complex or an RNA polymerizing complex comprising a nucleic acid polymerizing enzyme and a nucleic acid molecule.In a specific embodiment, the biomolecule is a DNA polymerase with a DNA binding cleft, particularly a family A DNA polymerase, including but not limited to Klenow, Taq, or T7 DNA polymerase, or any genetically modified form thereof, or a family B polymerase, including but not limited to Therminator, Phi29, RB-69, or T4 DNA polymerase, or any genetically modified form thereof.In this context, reference is made to US 7,745,116 B2, the contents of which are incorporated herein by reference.

[0037] In a further embodiment, the biomolecule is a nuclease, in particular an exonuclease, or a nucleic acid-degrading molecular complex, in particular a DNA-degrading molecular complex or an RNA-degrading molecular complex comprising a nuclease and a nucleic acid molecule.

[0038] In further embodiments, the biological molecule is a gene editing enzyme, in particular a Cas nuclease such as a Cas3, Cas9, Cas10, or Cas12 nuclease, or any genetically modified form thereof, e.g., a Cas nickase, or a gene editing complex comprising a gene editing enzyme and a nucleic acid molecule, e.g., a guide RNA and / or a target nucleic acid.

[0039] The single molecule event to be detected occurs in a sample containing components required for the event, e.g., biomolecules and, optionally, small molecules. At least one of the components is a luminescent component that includes a luminescent group, i.e., a group that can emit radiation in response to irradiation with excitation radiation. In some embodiments, the luminescent component is a compound that carries a luminescent labeling group. In some embodiments, the luminescent component is a compound that can itself emit light.

[0040] In certain embodiments, the luminescent component of a single-molecule event can be a reactant, a reaction intermediate, and / or a reaction product. In certain embodiments, the luminescent component is a fluorescent component, i.e., a component that can emit fluorescent radiation in response to being irradiated with excitation radiation. In certain embodiments, the fluorescent component is a compound that carries a fluorescent labeling group. In certain embodiments, the fluorescent component is a compound that can itself fluoresce.

[0041] In certain embodiments, a sample of a single molecule event comprises a plurality of different luminescent components, where at least some of the luminescent components have overlapping spectra of emitted radiation. For example, a sample may comprise a plurality of different fluorescent components, where at least some of the fluorescent components have distinguishable and overlapping fluorescence emission spectra.

[0042] The at least one luminescent component may be present in the sample in immobilized and / or free form. In certain embodiments, the at least one luminescent component is present in free form.

[0043] According to the present disclosure, the excitation radiation is split into a plurality of individual radiation beams by a diffractive optical element, hi certain embodiments, the excitation radiation is split into about 10 to about 1000, about 50 to about 500, and for example, about 100 individual radiation beams.

[0044] In some embodiments, the optical diffractive element is an optical diffraction grating, for example, a three-dimensional optical diffraction grating applied to an optically transparent carrier as needed. Light passing through the optical diffractive element is split, thereby generating a predetermined diffraction pattern of multiple radiation beams, which can form a desired arrangement of multiple optical foci in the structure plane through constructive and destructive interference. The production of suitable diffractive optical elements is described, for example, in F. Nikolaef's paper at the Chalmers Institute of Technologies (1999), M. Johansson's paper at the Chalmers Institute of Technologies (2001), and Johansson and Hard's publication (Applied Optics 38 (1999), 1302-1310). Suitable materials for producing optical elements are plastics, glass, and composite materials, or other materials that are optically transparent at a specified wavelength and can be processed by photolithographic etching.

[0045] Individual radiation beams of excitation radiation can be directed at an individual sample or a group comprising multiple individual samples. In certain embodiments, the group of samples comprises at most about 10 6 individual samples, and particularly from about 1,000 to about 10 6 , or about 5,000 to about 10 5 , for example, containing about 10,000 individual samples.

[0046] In certain embodiments, step (a) of the above method comprises focusing the multiple individual radiation beams generated by the diffractive optical element onto the multiple samples. In further embodiments, step (a) comprises directing the multiple individual radiation beams generated by the diffractive optical element onto the multiple samples without focusing them, for example by using one or more highly spatially coherent lasers to generate the excitation radiation.

[0047] In some embodiments, the multiple beams of excitation radiation generated by the optical diffractive element are passed to the sample through an optically transparent substrate of the support on which the sample is placed. In certain embodiments, the sample is placed on a surface of the support distal to the surface of the support through which the multiple beams of excitation radiation enter. In such embodiments, the individual beams of excitation radiation generate individual evanescent fields on the surface of the support on which the sample is placed. The individual evanescent fields can be generated via total internal reflection. In certain embodiments, the individual evanescent fields can have diameters of about 1 nm to about 100 μm, or about 10 nm to about 10 μm. In certain embodiments, the individual evanescent fields can cover up to about 100,000 individual samples, and particularly about 1,000 to about 100,000, or about 5,000 to about 20,000, e.g., about 10,000 individual samples.

[0048] In certain embodiments, multiple individual evanescent fields are generated. These individual evanescent fields may or may not overlap. In some embodiments, the individual evanescent fields do not substantially overlap. In some embodiments, the overlap between two adjacent evanescent fields is about 20% (by area) or less, or about 10% (by area) or less.

[0049] In certain embodiments, the detection means comprises a detector matrix comprising a plurality of detection pixels. The detection pixels can be optically projected onto the sample spot on the surface of the support. In certain embodiments, the detector provides an optical focus on each sample spot to achieve a desired precision in measuring single molecule events occurring at the sample spot. An essential part of the innovation is the creation of many individual evanescent fields of excitation energy from splitting a single laser beam by a diffractive optical element.

[0050] In certain embodiments, at least one reaction space is provided around said plurality of samples, wherein said reaction space comprises a medium, for example a liquid medium, and in particular an aqueous liquid medium, and components for said single molecule event.

[0051] In certain embodiments, the reaction space is a flow cell, which provides a flow of fresh components to the sample spot and a flow of used components from the sample spot. In certain embodiments, a single reaction space is provided around multiple samples. In further embodiments, individual reaction spaces are provided around each sample or around groups of samples. For example, the reaction space has a depth of about 10 nm to about 1,000 μm.

[0052] In certain embodiments, the path of the emitted radiation is from the sample spot through the reaction space to the detection means.

[0053] In a further specific embodiment, the radiation source is positioned above an optically transparent support that contains multiple sample spots at its lower surface, i.e., the surface distal to the radiation source. The lower surface of the support containing the sample spots is in contact with a single reaction space, which may be the above-mentioned flow cell, for example. The radiation source emits excitation radiation downward toward the support. The excitation radiation is split into multiple individual beams and then enters the support at its upper surface, i.e., the surface proximal to the radiation source. The individual radiation beams are reflected by the lower surface and thereby generate individual evanescent fields at the above-mentioned sample spots or groups of sample spots, for example, by total internal reflection. The path of the emitted radiation from the sample spots is directed downward through the reaction space to the detection means.

[0054] In some embodiments, the method of the present disclosure includes analyzing multiple consecutive single molecule events in one sample, and particularly includes analyzing multiple consecutive single molecule events separately in parallel in different samples. The term "multiple single molecule events" encompasses a series of consecutive single molecule events occurring in the same sample, for example, at sample spots on a support. In some embodiments, the series of consecutive single molecule events includes up to 10, up to 100, up to 1,000, up to 10,000, or even more individual single molecule events, for example, up to about 10,000,000 or 100,000,000 individual single molecule events. For example, the multiple single molecule events can include consecutive nucleic acid extension and / or nucleic acid degradation steps of single molecule nucleic acid sequence analysis.

[0055] In certain embodiments, single molecule nucleic acid sequencing involves multiple steps of nucleic acid extension, in which a light-emitting nucleotide building block, e.g., a light-emitting labeled nucleoside polyphosphate containing 3 to 15 phosphate groups, is incorporated into a nucleic acid molecule in the presence of a nucleic acid polymerizing enzyme, e.g., a DNA polymerase or an RNA polymerase. These embodiments can include detecting emitted radiation from the incorporation of the light-emitting nucleotide building block, e.g., a light-emitting labeled nucleoside polyphosphate, into the nucleic acid molecule.

[0056] The present disclosure further provides an apparatus for analyzing single molecule events, said apparatus comprising: - means for providing a plurality of samples configured for single molecule events; - an optical excitation device including a radiation source configured to emit excitation radiation, a diffractive optical element for splitting the excitation radiation into a plurality of individual radiation beams, and an optical arrangement for directing the plurality of individual radiation beams towards the plurality of samples; - detection means configured to capture emitted radiation from said plurality of samples; and - analyzing means configured to analyze the captured emitted radiation; wherein the paths of excitation radiation from the radiation source to the plurality of samples and the paths of emission radiation from the plurality of samples to the detection means do not overlap.

[0057] In some embodiments, the device is configured to perform the methods described above, for example, to perform single molecule nucleic acid sequencing.

[0058] The disclosed methods and devices are useful for providing high-throughput analysis of single molecule events. In one embodiment, high-throughput analysis is performed at a rate of about 10 6 from about 10 11 Parallel analysis of individual samples, particularly on a single support, e.g., about 10 6 from about 10 11 In some embodiments, the high-throughput analysis is performed in a single reaction space.

[0059] Methods and apparatus for analyzing single molecule events are disclosed, for example, in WO2002 / 097406, WO2003 / 052137, WO2006 / 013110, WO2013 / 131888, WO2015 / 104245, WO2017 / 001407, and WO2018 / 104301, the contents of which are incorporated herein by reference.

[0060] For the analysis of single molecule events, biomolecules can be placed in sample spots on the support, where they are contacted with a sample solution containing free reaction partners, thereby defining one or more reaction spaces. In particular, at least 100, at least 1000, or at least 10,000, and up to 10 6 Many more molecules can be analyzed on a single support, for example, on a single planar support.

[0061] The nucleic acid molecules to be sequenced can be selected from DNA molecules, such as genomic DNA fragments, cDNA molecules, plasmids, or RNA molecules, such as mRNA molecules. The nucleic acid molecules can be derived from a genomic or expression library generated from a cell or organism, e.g., a eukaryotic or prokaryotic cell or organism. This can be achieved by using a plurality of different nucleic acid template molecules, e.g., at least 10, 100, 1,000, or 10,000, and up to 100,000, 10 6 , or 10 7 This allows for the parallel sequencing of , or even more, different nucleic acid molecules.

[0062] The nucleic acid molecule to be sequenced can be a single-stranded nucleic acid molecule in linear or circular form, for example, in covalently linked circular form.To obtain circular nucleic acid template, linear nucleic acid molecule can be subjected to circularization procedure and optionally strand separation procedure during sample preparation.Circularization can be achieved by ligation according to known protocols, for example, using DNA ligase or RNA ligase.In some embodiments, adapter molecule and / or identifier molecule, i.e., nucleic acid molecule of known sequence, can be attached to nucleic acid molecule.

[0063] Sequencing can involve nucleic acid extension and / or nucleic acid degradation. The sequencing process involves one or more sequencing cycles.

[0064] The nucleic acid synthesizing enzyme molecule can extend a primer annealed to a nucleic acid template molecule. The primer extension can be carried out by stepwise incorporating individual nucleotide building blocks into the 3'-end of the growing nucleic acid chain, thereby generating a nucleic acid molecule complementary to the sequence of the circular nucleic acid template. The nucleic acid synthesizing enzyme is selected from polymerases capable of template-specific nucleic acid polymerization, preferably from natural or modified polymerases, including DNA polymerases and RNA polymerases, such as thermostable DNA polymerases.

[0065] Specific examples of suitable DNA polymerases include Taq polymerase, exonuclease-deficient Taq polymerase, E. coli DNA polymerase I, Klenow fragment, reverse transcriptase, Φ29-related polymerases, including wild-type Φ29 polymerase, and derivatives of such polymerases, such as exonuclease-deficient versions, T7 DNA polymerase, T5 DNA polymerase, RB69 polymerase, and the like.

[0066] Nuclease molecules can gradually separate individual nucleotide building blocks from nucleic acid molecules.Preferably, exonucleases are used, more preferably single-stranded exonucleases that degrade in the 3'→5' or 5'→3' direction.Particularly preferred exonucleases are 3'→5' exonucleases such as E. coli exonuclease I and E. coli exonuclease III, and 5'→3' exonucleases such as T7 exonuclease, E. coli exonuclease II, and E. coli exonuclease VIII.In addition, the exonuclease activity of various polymerases, such as Klenow fragment, Taq polymerase, or T4 polymerase, can be used.

[0067] The nucleic acid synthesizing enzyme molecule is contacted with a linear or circular nucleic acid template molecule, such as a single-stranded DNA or RNA molecule, and a primer molecule that is annealed to or can anneal to the nucleic acid template molecule. The primer molecule is preferably a single-stranded nucleic acid or nucleic acid analog molecule with a free 3' end that can be extended by an enzymatic reaction catalyzed by the immobilized nucleic acid synthesizing enzyme molecule. The length of the primer molecule is selected to enable effective annealing to the template under reaction conditions. Typically, the length of the primer molecule is at least 8, at least 10, at least 12, or at least 15 nucleotides, and for example, up to 20, 25, 50, or 100 nucleotides, or even longer. In some embodiments, the primer is resistant to digestion by the nuclease molecule, for example, by incorporating a nucleotide analog building block and / or an analog bond between the nucleotide building blocks that is stable against degradation. In other embodiments, the primer is susceptible to digestion by the nuclease molecule.

[0068] The sequence of the primer is selected so that it can effectively anneal to the template molecule under the reaction conditions. For example, the primer can be a universal degenerate primer that can statistically anneal to unknown nucleic acid sequences. In other embodiments, the primer can be capable of annealing to a known sequence portion of the nucleic acid template molecule. In this embodiment, a known adapter and / or identifier sequence can be incorporated into the nucleic acid template molecule. The primer can be unlabeled or can contain a fluorescent labeling group.

[0069] Furthermore, the presence of luminescent nucleotide building blocks, e.g., nucleotide building blocks carrying at least one fluorescent labeling group, is essential, and preferably each different nucleotide building block (A, G, C, T / U) contains a different fluorescent labeling group.

[0070] The fluorescent labeling group may be selected from known fluorescent labeling groups used for labeling biopolymers, particularly nucleic acids, such as, for example, fluorescein dyes, rhodamine, oxazines such as Evoblue or Gnothis Blue, phycoerythrin, Cy3, Cy5, IR dyes, or derivatives thereof.

[0071] A nucleotide building block may carry (i) a fluorescent labeling group that remains on the building block when the building block is incorporated into a nucleic acid molecule during primer extension catalyzed by a nucleic acid synthesizing enzyme molecule, and / or (ii) a fluorescent labeling group that is cleaved from the building block when the building block is incorporated into a nucleic acid molecule during primer extension catalyzed by a nucleic acid synthesizing enzyme molecule. The fluorescent labeling group that remains on the building block is preferably attached to the α-phosphate group, sugar, and / or nucleobase.

[0072] In certain embodiments, the fluorescent labeling group remaining in the building block is attached to the nucleobase, e.g., via a linker that may have a chain length of up to 15, preferably 10-12, carbon atoms, optionally including a heteroatom, e.g., N, O, or S atom. The fluorescent labeling group that is cleaved upon incorporation of the building block into a nucleic acid molecule may be attached, for example, to the terminal phosphate group of a polyphosphate building block, including, but not limited to, a hexa-, penta-, tetra-, or triphosphate building block, e.g., the γ-phosphate group of a triphosphate building block. In certain embodiments, a building block is selected that contains both (i) a fluorescent labeling group that remains after incorporation and (ii) a fluorescent labeling group that is cleaved during incorporation. In this case, fluorescent groups that can interact with each other by, for example, quenching and / or energy transfer, may be selected.

[0073] The nucleic acid molecule to be sequenced contains a fluorescent labeling group if the nucleic acid molecule is subjected to direct sequencing using a nuclease molecule, whereas the nucleic acid molecule to be sequenced may not contain a fluorescent labeling group if the nucleic acid molecule is used as a template in primer extension.

[0074] The sequencing procedure may include a step of generating a nucleic acid molecule having incorporated nucleotide building blocks in primer extension catalyzed by a nucleic acid polymerase molecule, and / or a second step of cleavage of individual nucleotide building blocks from the generated nucleic acid molecule catalyzed by a nuclease molecule. Depending on the type of fluorescent label, nucleic acid sequencing may be performed during primer extension and / or degradation.

[0075] Sequencing during primer extension involves the use of nucleotide building blocks that carry a fluorescent labeling group that is cleaved from the building block when the nucleotide building block is incorporated into a nucleic acid molecule. In this case, the time-dependent fluorescence change caused by the cleavage of the fluorescent labeling group from the nucleotide building block can be determined. Sequencing during nucleic acid degradation involves the use of nucleotide building blocks that carry a fluorescent labeling group that remains on the building block when the building block is incorporated into a nucleic acid molecule. The gradual cleavage of individual nucleotide building blocks from the nucleic acid molecule causes a time-dependent change in fluorescence as the labeled nucleotide building block is released from the nucleic acid molecule. In some embodiments, performing sequencing during extension and degradation is also possible, i.e., when using nucleotide building blocks that carry both a fluorescent labeling group that remains on the building block and a fluorescent labeling group that is cleaved from the building block when the building block is incorporated into a nucleic acid molecule. In this embodiment, both fluorescent groups can be the same or different.

[0076] In some embodiments, the method comprises one or more cycles of nucleic acid synthesis and nucleic acid degradation to determine the base sequence of a nucleic acid molecule template. Nucleic acid synthesis comprises the extension of a primer annealed to the nucleic acid template molecule, catalyzed by a nucleic acid polymerase molecule, whereby a nucleic acid molecule complementary to the sequence of the nucleic acid template is generated. In the next step, the generated nucleic acid molecule is degraded by a nuclease molecule.

[0077] When a nucleotide building block is incorporated into an extended nucleic acid molecule, a time-dependent change in fluorescence can occur, which can be detected as described above. Preferably, incorporation of a nucleotide building block into an extended nucleic acid molecule is associated with a detectable increase in fluorescence, preferably a transient increase in fluorescence. For example, a nucleotide building block can be used that carries a fluorescent labeling group, for example, on the γ-phosphate, in the portion of the molecule that is cleaved when the building block is incorporated into the primer.

[0078] When nucleotide building blocks are cleaved from the synthesized nucleic acid molecule, the fluorescent labeling group incorporated into the nucleic acid chain interacts with, for example, chemical groups in the nucleic acid, particularly nucleic acid bases such as G, or / and adjacent fluorescent labeling groups, resulting in a change in fluorescence, particularly in fluorescence intensity, due to quenching or / and energy transfer processes, compared to the so-called "isolated" form of the fluorescent labeling group, and this time-dependent change in fluorescence can be measured. The cleavage and removal of individual nucleotide building blocks changes the overall fluorescence, for example, the fluorescence intensity of the immobilized nucleic acid chain, and this change is a function of the cleavage and removal of individual nucleotide building blocks, i.e., a function of time.

[0079] In certain embodiments, the association of the labeled nucleotide with the biomolecular complex is detected by measuring the polarization of the emitted photons. The polarization of the excited state photons is altered by the rotational motion of the light-emitting nucleotide label and can be used to identify the free-floating, unbound labeled nucleotides in the polymerization process.

[0080] This time-dependent change in fluorescence during extension and / or degradation can be recorded in parallel for multiple nucleic acid molecules and correlated with the base sequence of each nucleic acid strand. Preferably, fluorescent labeling groups are used that are at least partially quenched when incorporated into a nucleic acid strand, so that the fluorescence intensity increases after the nucleotide unit containing the labeling group or an adjacent unit that causes quenching is removed by cleavage.

[0081] During the incorporation and / or removal of individual nucleotide building blocks, it is possible to measure the change in fluorescence intensity of the nucleic acid strand and / or of the incorporated or cleaved nucleotide building blocks due to quenching or energy transfer processes. This change in fluorescence intensity over time depends on the base sequence of the nucleic acid strand being studied and can therefore be correlated with said sequence.

[0082] The complete sequence of a nucleic acid molecule can be determined by using a mixture of nucleotide building blocks labeled with all four different bases, e.g., A, G, C, and T, or a mixture of nucleotide building blocks labeled with combinations of two or three different bases. If appropriate, a "sequence identifier," i.e., a labeled nucleic acid of known sequence, can be attached to the nucleic acid strand being studied, e.g., by enzymatic reaction using ligase and / or terminal transferase, so that a known fluorescence pattern is initially obtained at the start of sequencing, followed by a fluorescence pattern corresponding to the unknown sequence being studied.

[0083] Detection involves irradiating the support with excitation radiation from a radiation source, preferably by means of a laser or another suitable light source, to cause excitation of the fluorescent labeling group. In certain embodiments, the radiation source comprises a plurality of different lasers emitting radiation at different wavelengths. In this regard, it is possible to use one or more laser beams, for example, an expanded laser beam having a cross section of about 1 to 20 mm, and / or multiple laser beams. Detection preferably involves multipoint fluorescence excitation by laser, for example, a dot matrix of laser dots generated via diffractive optics (see WO 2002 / 097406) or a quantum well laser.

[0084] The fluorescence emission of multiple nucleic acid strands can be detected in parallel using a detection means such as an electronic detector matrix, for example, a CCD camera, a CMOS detector matrix, or a detector matrix including an avalanche photodiode matrix. Detection can be performed in such a way that fluorescence excitation and detection are performed in parallel for some or all of the nucleic acid strands being studied. Preferably, detection is performed on fluorescent light emitted from the support surface in an essentially perpendicular direction through the reaction space or support.

[0085] Detection can be performed, for example, by means of single molecule detection, for example, by fluorescence correlation spectroscopy. -21 From 10 -10 The method involves exposing a very small, preferably confocal, volume element of about 1 μm to excitation light from a laser or another suitable light source. This excitation light excites receptors present in the measurement volume, causing them to fluoresce. The fluorescence emitted from the measurement volume is measured by a photodetector, and the measured change in emission over time is correlated with the concentration of the analyte, thereby making it possible to identify individual molecules in the measurement volume at suitable high dilutions. Details of the procedure and the equipment used for detection can be found in the disclosure of EP 0 679 251, the contents of which are incorporated herein by reference. Confocal determination of single molecules is further described in Rigler and Mets (Soc. Photo-Opt. Instrum. Eng. 1921 (1993), 239 ff.) and Mets and Rigler (J. Fluoresc. 4 (1994) 259-264), the contents of which are incorporated herein by reference.

[0086] Alternatively or additionally, detection can also be performed by a method of time-resolved decay measurement called "time gating," as described, for example, by Rigler et al., "Picosecond Single Photon Fluorescence Spectroscopy of Nucleic Acids," in "Ultrafast Phenomena," D.H. Australianton, Ed., Springer 1984, the contents of which are incorporated herein by reference. Here, fluorescent molecules are excited in the measurement volume, followed by a detection interval of the photodetector, e.g., a time interval of ≥ 100 ps. In this way, the background signal generated by the Raman effect can be kept sufficiently low, allowing for the detection of single molecules in an essentially interference-free manner.

[0087] The disclosed methods and apparatus are also suitable for the analysis of additional multiple single molecule events, where there is a high demand for high-yield single molecule analysis, i.e., the analysis of multiple single biomolecules binding to multiple spots selected for multiple analysis.

[0088] In certain embodiments, the present disclosure relates to single molecule analysis of receptor-ligand interactions, including, for example, binding of a receptor protein to a sample spot and subsequent study of its interaction with its ligand, for example, in drug development.

[0089] In further embodiments, the present disclosure relates to single molecule analysis of hybridization events, including the attachment of short single-stranded nucleic acid molecules, such as DNA or RNA molecules, having lengths in the range of 3-300 nucleotides, followed by the addition of a sample containing a complementary nucleic acid molecule and the observation of any hybridization events. Applications can include, for example, viral RNA / DNA detection, bacterial DNA / RNA detection, and the detection of short DNA fragments from cancer cells in the bloodstream.

[0090] The present disclosure will be further described in detail by reference to the following specific embodiments. [Example]

[0091] Figure 1 shows an embodiment of the present disclosure. A laser beam (12) is split into multiple individual beams, e.g., four individual beams (16), by a diffractive optical element (14). The individual beams (14) are directed toward a sample spot (18) disposed on a support (20), where the individual beams (14) generate a predetermined pattern of optical foci. Light (22) emitted from the foci at the sample spot (18) is directed toward a detector (26) by an optical arrangement (24). The difference between this embodiment and the prior art embodiment (previous slide) is that the optical path of the radiation source and the optical path of the emitted light are not the same throughout their paths and do not overlap.

[0092] Figure 2 shows a further embodiment of the present disclosure in which detection is based on evanescent fields. In Figure 2, a single evanescent field is depicted, for example, one of a total of 10,000 evanescent fields.

[0093] Individual incident light beams (30), generated by passing excitation light through an optical diffraction element (not shown), pass through a gaseous medium (32) onto an optically transparent support (34), e.g., made of silicon dioxide. They enter the support (34) at a surface (34a) distal to a surface (34b) on which a sample spot (36) is located. Binding molecules (38) that mediate immobilization of single biomolecules or single-molecule complexes (40), e.g., DNA or RNA polymerase, or complexes containing DNA or RNA polymerase, a nucleic acid template, and a primer, are attached to the surface of the sample spot (36). The sample spot (36) is surrounded by a reaction space (42) containing a medium, e.g., an aqueous medium, and components necessary for single-molecule events, e.g., fluorescently labeled nucleotide building blocks. The incident light beams (30) are reflected from the surface (34b), generating a totally reflected light (44) and an evanescent field (46). The evanescent field (46) can encompass a single sample spot (36) or, as shown here, a collection of sample spots (36), e.g., up to 100,000, and typically about 10,000, sample spots, and can have diameters of, e.g., 1 nm to 100 μm. Because the excitation light is split into multiple individual beams, multiple evanescent fields can be generated at the support surface (34b). Emission light from single-molecule events occurring on the sample spots (36) is passed to a detector (not shown).

[0094] 3 illustrates a further embodiment of the present disclosure. A support (50) is provided having a sample spot (52) on its surface (not shown) with a single molecule immobilized thereon. The sample spot (52) is in contact with a single reaction space (54), which is a flow cell including an inlet (56) for the inflow of a medium containing fresh components for interaction with the single molecule, and an outlet (58) for the outflow of a medium containing spent components. Emitted light (not shown) from the sample spot (52) passes through the reaction space (54) to a detector (60). The detector includes pixels that are optically projected onto the sample spot (52) on the surface of the support (50).

Claims

1. 1. A method for analyzing single molecule events, the method comprising: (a) providing a plurality of samples in a field where single molecule events occur; wherein said plurality of samples are present on a support, wherein said support comprises an optically transparent substrate and a plurality of sample spots on a surface of the support, wherein the sample spots are spatially separated from one another, and wherein said samples are present on said sample spots; and providing a plurality of samples to a site where single molecule events occur, wherein a component of said single molecule event is immobilized on at least one sample spot, and wherein said immobilized component comprises a single biomolecule; (b) illuminating the plurality of samples with an optical excitation device including a radiation source, a diffractive optical element for splitting excitation radiation into a plurality of individual radiation beams, and an optical arrangement for directing the plurality of individual radiation beams toward the plurality of samples; (c) capturing radiation emitted from said plurality of samples with a detection means; and (d) analyzing the captured emitted radiation; Including, wherein a path of excitation radiation from the radiation source to the plurality of samples and a path of emission radiation from the plurality of samples to the detection means do not overlap; wherein the plurality of beams generated by the optical diffractive element are passed through an optically transparent substrate of a support to the plurality of samples, wherein the plurality of samples are positioned on a surface of the support distal to a surface of the support through which the excitation radiation enters; wherein each radiation beam of excitation radiation generates, by total internal reflection, a respective evanescent field on the surface of a support on which the plurality of samples are disposed; and Here, each individual evanescent field illuminates a cluster of sample spots, The method.

2. 2. The method of claim 1, wherein the path of excitation radiation from said radiation source originates from a plane above the path of radiation from said plurality of samples to said detection means.

3. Each radiation beam of excitation radiation is directed at a group containing a plurality of individual samples, wherein the group is at most about 10 ６ individual samples, and in particular from about 1,000 to about 10 ６ , or from about 5,000 to about 10 ５ 10. The method of any one of the preceding claims, comprising, for example, about 10,000 individual samples.

4. the individual evanescent fields have a diameter of about 1 nm to about 100 μm, or about 10 nm to about 10 μm, and wherein the individual evanescent fields have a diameter of at most about 10 ６ individual samples, and in particular from about 1,000 to about 10 ６ of, or from about 5,000 to about 10 ５ 10. The method of any one of the preceding claims, covering, for example, about 10,000 individual samples.

5. 10. The method of any one of the preceding claims, wherein said detection means comprises a detector matrix comprising a plurality of detection pixels, wherein said detection pixels are optically projected onto sample spots on the surface of the support.

6. 10. The method of any one of the preceding claims, wherein at least one reaction space is provided around said plurality of samples, wherein said reaction space comprises a medium and components for said single molecule event.

7. The method of claim 6 , wherein the path of the emitted radiation is through the reaction space.

8. 10. The method of any one of the preceding claims, wherein said single molecule event comprises sequencing of a single nucleic acid molecule.

9. 1. An apparatus for analyzing single molecule events, said apparatus comprising: - a means for providing a plurality of samples configured for single molecule events, wherein said plurality of samples are present on a support, wherein said support comprises an optically transparent substrate and a plurality of sample spots on a surface of the support, wherein the sample spots are spatially separated from one another, and wherein said samples are present on said sample spots; and means for providing a plurality of samples configured for single molecule events, wherein a component of said single molecule events is immobilized on at least one sample spot, and wherein said immobilized component comprises a single biomolecule; an optical excitation device comprising a radiation source configured to emit excitation radiation, a diffractive optical element for splitting said excitation radiation into a plurality of individual radiation beams, and an optical arrangement for directing said plurality of individual radiation beams towards said plurality of samples; - detection means adapted to capture emitted radiation from said plurality of samples; and - analysis means adapted to analyze the captured emitted radiation; Including, wherein a path of excitation radiation from the radiation source to the plurality of samples and a path of emission radiation from the plurality of samples to the detection means do not overlap; wherein the apparatus is configured to pass the plurality of beams generated by the optical diffractive element through an optically transparent substrate of a support to the plurality of samples, wherein the plurality of samples are positioned on a surface of the support distal to a surface of the support through which the excitation radiation enters; wherein each radiation beam of excitation radiation generates, by total internal reflection, each evanescent field on the surface of a support on which the plurality of samples are disposed; and Here, each individual evanescent field illuminates a cluster of sample spots, The device.

10. 10. The apparatus of claim 9 configured to perform single molecule nucleic acid sequencing.

11. Use of the method of any one of claims 1 to 8 or the device of any one of claims 9 to 10 to provide high-throughput analysis of single molecule events.

12. High-throughput analysis can be performed on approximately 10 ６ from about 10 １１ 12. Use according to claim 11, comprising parallel analysis of individual sample spots of

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