High throughput analysis of single molecule events

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to the analysis of single molecule events as observed by irradiating samples with excitation radiation and detecting emission radiation from the samples induced by the excitation radiation, wherein the excitation radiation path from the radiation source to the multiple samples does not overlap with the emission radiation path from the multiple samples to the detection device. Background Art

[0002] The sequencing of the human genome or the genomes of other organisms and the determination and comparison of individual sequence variants requires the provision of sequencing methods which are firstly rapid and secondly can be used routinely and cost-effectively.

[0003] The high demand for cost-effective order-checking has promoted the development of high-throughput sequencing technology, and described technology makes the sequencing process parallelization that produces multiple sequences simultaneously.The example of these order-checking technologies is large-scale parallel feature order-checking (LynxTherapeutics), clone amplification order-checking (Life Technologies), 454 pyrophosphate order-checking (RocheDiagnostics), illumina order-checking (SolexaInc.), connection method order-checking (sequencing byligation) (LifeTechnologies), ion semiconductor order-checking (Life Technologies) or DNA nanometer ball order-checking (CompleteGenomics).These technologies allow the consensus sequence in rapid analysis nucleic acid colony.But the sudden change that is present in the minority sequence in nucleic acid colony to be analyzed, for example, the sudden change that is present in minority cell genome will not be detected, because they are covered by most of other sequences that exist in colony.

[0004] In order to address these problems, different forms of single molecule analysis have been developed. Several methods in these methods belong to the field of fluorescence spectroscopy (FCS), and relate to the detection and analysis of single molecules by fluorescence. Generally, in the case of DNA, DNA nucleic acid polymerase and / or nucleic acid degrading enzyme and fluorescently labeled nucleic acid and / or nucleotide building blocks are used to measure the sequence of a single nucleic acid molecule separately, and the sequence determination is based on the time-dependent variation of fluorescence when the nucleotide building blocks are incorporated into nucleic acid molecules or excised from nucleic acid molecules. Single molecule sequencing methods and the equipment suitable for carrying out such methods are for example described in jointly owned applications WO 2002 / 097406, WO 2003 / 052137, WO 2006 / 013110, WO 2013 / 131888, WO 2015 / 104245, WO 2017 / 001407 and WO 2018 / 104301.

[0005] Besides DNA and / or RNA sequencing, there are many additional applications for which there is a high demand for high-throughput single-molecule analysis.

[0006] US 7,259,847 B2 (the disclosure of which is incorporated by reference) discloses a method for measuring luminescent molecules by optical excitation in a confocal measurement volume, the method comprising the following steps:

[0007] (a) providing a sample comprising a luminescent molecule,

[0008] (b) illuminating the sample with an optical excitation device comprising a light source, a diffractive optical element for splitting the passing light into a plurality of focal points, and a focusing optical arrangement for focusing the passing plurality of light beams into a plurality of confocal volume elements, and

[0009] (c) Capturing the emitted radiation from multiple confocal volume cells.

[0010] No embodiments are disclosed in which the excitation radiation path and the emission radiation path do not overlap.

[0011] One of the several objects of the present disclosure is to provide methods and apparatus for high-throughput analysis of single molecule events.

[0012] Summary of the Invention

[0013] In a first aspect, the present disclosure relates to a method for analyzing single molecule events, comprising:

[0014] (a) providing a plurality of samples in which a single molecular event occurs,

[0015] wherein the plurality of samples are present on a carrier, wherein the carrier comprises an optically transparent substrate and a plurality of sample spots on a surface of the carrier, wherein the sample spots are spatially separated from each other, and wherein the samples are present on the sample spots, and

[0016] wherein a component of the single molecule event is immobilized to at least one sample site, and wherein the immobilized component comprises a single biomolecule,

[0017] (b) irradiating the plurality of samples with an optical excitation device comprising a radiation source, 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 to the plurality of samples,

[0018] (c) capturing the emitted radiation from the plurality of samples by a detection device, and

[0019] (d) analyzing the captured emitted radiation, wherein a path of the excitation radiation from the radiation source to the plurality of samples does not overlap with a path of the emitted radiation from the plurality of samples to the detection device, wherein the plurality of light beams generated by the optical diffraction element are transferred to the plurality of samples through an optically transparent substrate of a carrier, wherein the plurality of samples are located on a surface of the carrier that is distal to a surface of the carrier through which the excitation radiation enters the carrier,

[0020] wherein the individual beams of excitation radiation generate individual evanescent fields on the carrier surface where the plurality of samples are located by total internal reflection, and

[0021] Each individual evanescent field illuminates a set of sample points.

[0022] In certain embodiments, a single molecule event comprises sequence analysis of a single nucleic acid molecule.

[0023] Another aspect relates to an apparatus for analyzing single molecule events, comprising:

[0024] - means for providing a plurality of samples adapted for single molecule events,

[0025] wherein the plurality of samples are present on a carrier, wherein the carrier comprises an optically transparent substrate and a plurality of sample spots on a surface of the carrier, wherein the sample spots are spatially separated from each other, and wherein the samples are present on the sample spots, and

[0026] wherein a component of the single molecule event is immobilized to at least one sample site, and wherein the immobilized component comprises a single biomolecule,

[0027] an optical excitation device comprising a radiation source adapted to emit excitation radiation, a diffractive optical element for splitting said excitation radiation into a plurality of individual radiation beams and optical means for directing (e.g. focusing) said plurality of individual radiation beams to said plurality of samples,

[0028] - detection means adapted to capture emitted radiation from said plurality of samples, and

[0029] - analyzing means adapted to analyze the captured emitted radiation,

[0030] wherein an excitation radiation path from the radiation source to the plurality of samples does not overlap with an emission radiation path from the plurality of samples to the detection device,

[0031] wherein the apparatus is adapted to pass the plurality of light beams generated by the optical diffraction element through an optically transparent substrate of a carrier to the plurality of samples, wherein the plurality of samples are located on a surface of the carrier remote from a carrier surface through which the excitation radiation enters the carrier.

[0032] wherein the individual beams of excitation radiation generate individual evanescent fields on the carrier surface where the plurality of samples are located by total internal reflection, and

[0033] Each individual evanescent field illuminates a set of sample points.

[0034] In certain embodiments, the device is suitable for sequence analysis of single nucleic acid molecules.

[0035] Another aspect relates to the use of the above method or the above device for providing high-throughput analysis of single molecule events.

[0036] Items in the instruction manual

[0037] 1. A method for analyzing single molecule events, comprising:

[0038] (a) providing a plurality of samples in which a single molecular event occurs,

[0039] (b) irradiating the plurality of samples with an optical excitation device comprising a radiation source, 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 to the plurality of samples,

[0040] (c) capturing the emitted radiation from the plurality of samples by a detection device, and

[0041] (d) analyzing the captured emitted radiation,

[0042] The excitation radiation path from the radiation source to the plurality of samples does not overlap with the emission radiation path from the plurality of samples to the detection device.

[0043] 2. The method of item 1, wherein the excitation radiation path from the radiation source originates from a plane above the emission path from the plurality of samples to the detection device.

[0044] 3. The method of item 1 or 2, wherein the plurality of samples are present on a carrier.

[0045] 4. The method of item 3, wherein the carrier comprises a substrate and a plurality of sample spots on the surface of the carrier, wherein the sample spots are spatially separated from each other, and wherein the sample is present on the sample spots.

[0046] 5. The method of item 4, wherein the substrate is optically transparent.

[0047] 6. The method of item 4 or 5, wherein the components of the single molecule event are immobilized on the at least one sample site.

[0048] 7. The method of item 6, wherein the immobilized component comprises a single biological moiety, such as a single biological molecule.

[0049] 8. The method of any of the preceding items, wherein the radiation source comprises at least one laser.

[0050] 9. The method of any of the preceding items, wherein the plurality of samples comprises 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 A single sample.

[0051] 10. The method of any of the preceding items, wherein the individual radiation beams of the excitation radiation are split into about 10 to about 1000, or about 50 to about 500, such as about 100, individual radiation beams.

[0052] 11. The method of any of the preceding items, wherein individual radiation beams of excitation radiation are directed to individual samples or a group comprising a plurality of individual samples.

[0053] 12. The method of any of the preceding items, wherein the single beam of excitation radiation is directed to about 10 to about 10 6 or about 100 to about 10 5 A group consisting of multiple individual samples.

[0054] 13. The method of item 11 or 12, wherein a group comprises up to about 10 6 individual samples, particularly from about 1,000 to about 10 6 or about 5,000 to about 10 5 For example, about 10,000 individual samples.

[0055] 14. The method of any of the preceding items, wherein step (a) comprises focusing the plurality of individual radiation beams onto the plurality of samples.

[0056] 15. The method of any of the preceding items, wherein the plurality of light beams generated by the optical diffraction element are transferred to the plurality of samples through an optically transparent substrate of a carrier, the plurality of samples being located on the carrier.

[0057] 16. The method of item 15, wherein the plurality of samples are located on a carrier surface that is remote from the carrier surface through which the excitation radiation enters the carrier.

[0058] 17. The method of item 16, wherein the individual beams of excitation radiation generate individual evanescent fields on the carrier surface where the plurality of samples are located.

[0059] 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.

[0060] 19. The method of item 17 or 18, wherein the individual evanescent fields cover up to about 10 6 individual samples, particularly from about 1,000 to about 10 6 or about 5,000 to about 20,000, for example about 10,000, individual samples.

[0061] 20. The method of any one of items 17 to 19, wherein the separate evanescent fields are generated by total internal reflection.

[0062] 21. The method of any of items 17 to 20, wherein a plurality of separate evanescent fields are generated.

[0063] 22. The method of item 21, wherein the evanescent fields do not substantially overlap.

[0064] 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.

[0065] 24. The method of any of the preceding items, wherein the diffractive optical element is a grating.

[0066] 25. The method of any of the preceding items, wherein the detection device comprises a detector matrix comprising a plurality of detection pixels.

[0067] 26. The method of item 25, wherein the detection pixels are optically projected onto sample spots on the carrier surface.

[0068] 27. The method of any of the preceding items, wherein the detection device provides an optical focus on a single sample site.

[0069] 28. The method of any of the preceding items, 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 events.

[0070] 29. The method of item 28, wherein the reaction space is a flow cell.

[0071] 30. The method of item 28 or 29, wherein a single reaction space is provided around the plurality of samples.

[0072] 31. The method of any one of items 28 to 30, wherein the reaction space has a depth of about 10 nm to about 1000 μm.

[0073] 32. The method of any one of items 28 to 31, wherein the emitted radiation path passes through the reaction space.

[0074] 33. The method of any of the preceding items, wherein the single molecule event comprises sequence analysis of a single nucleic acid molecule.

[0075] 34. An apparatus for analyzing single molecule events, comprising:

[0076] - means for providing a plurality of samples adapted for single molecule events,

[0077] an optical excitation device comprising a radiation source adapted 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,

[0078] - detection means adapted to capture emitted radiation from said plurality of samples, and

[0079] - analyzing means adapted to analyze the captured emitted radiation,

[0080] The excitation radiation path from the radiation source to the plurality of samples does not overlap with the emission radiation path from the plurality of samples to the detection device.

[0081] 35. The apparatus of item 34, adapted to carry out the method of any one of items 1 to 33.

[0082] 36. The device of item 34 or 35, which is adapted to perform single molecule nucleic acid sequence analysis.

[0083] 37. Use of the method of any one of items 1 to 33 or the apparatus of any one of items 34 to 36 for providing high throughput analysis of single molecule events.

[0084] 38. The method of claim 37, wherein the high-throughput analysis comprises about 10 6 to about 10 11 Parallel analysis of individual samples.

[0085] 39. The use of item 38, wherein the high-throughput analysis comprises the analysis of about 10 6 to about 10 11 Parallel analysis of individual sample points.

[0086] 40. The use of item 38 or 39, wherein the high-throughput analysis is performed in a single reaction space.

[0087] Detailed description

[0088] The present disclosure provides methods and apparatus for analyzing single molecule events, wherein the single molecule event is 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 comprises a reaction of a biological moiety associated with the emission of characteristic electromagnetic radiation.

[0089] The present disclosure addresses the need for improved high-throughput methods in the analysis of single molecule events as observed by irradiating a sample with excitation radiation and simultaneously detecting the resulting emitted radiation induced by said irradiation from the sample. The concepts described in the present disclosure include the parallel analysis of multiple groups of individual sample spots on a single support, where each group may have up to 10 6 The diffractive optical element that splits the excitation radiation beam can generate many groups (e.g., up to 10 4 Up to 10 5 Thus, the total number of sample spots to be analyzed according to the present disclosure (particularly on a single carrier) may be about 10 6 Up to about 10 11 Furthermore, the present disclosure provides a reaction space containing a sample solution (eg, a liquid medium in contact with a sample point) that requires only a small volume, allowing for greater flexibility in sample solution distribution.

[0090] In particular, the present disclosure provides apparatus and methods for irradiating a plurality of samples with excitation radiation generated by a radiation source (e.g., a radiation source comprising at least one laser) and split by an optical diffraction element into a plurality of separate radiation beams, wherein the plurality of separate radiation beams are directed toward the plurality of samples. The samples contain components capable of emitting emission radiation, which is captured by a detection device and analyzed.

[0091] According to the present disclosure, the excitation radiation paths from the radiation source to the multiple samples and the emission radiation paths from the multiple samples to the detection device do not overlap. Therefore, a much larger surface area can be obtained on which the sample points are located, thereby greatly increasing the number of samples that can be analyzed.

[0092] In certain embodiments, the excitation radiation path from the radiation source originates from a plane above (ie, spatially above) the emission path from the plurality of samples to the detection device.

[0093] In certain embodiments, the plurality of samples comprises at least 10, at least 100, at least 1,000, at least 10 4 At least 10 5 or at least 106 and up to 10 8 10 9 Up to 10 10 or up to 10 11 A single sample.

[0094] The sample is typically present on a carrier. The carrier may comprise a substrate and a plurality of sample sites on the carrier surface, the sample sites being spatially separated from one another. In certain embodiments, the carrier surface is formed by the substrate and the sample sites, and wherein the sample is present on the sample sites. In certain embodiments, the substrate forms a continuous region over which the sample sites are distributed. A single sample site on the carrier surface is surrounded by a substrate that is, for example, different in material and / or surface from the sample site. Typically, the substrate is adapted to inhibit and / or block adhesion of biomolecules such as polypeptides, while the sample sites are adapted to allow adhesion of the desired biomolecules.

[0095] At the sample site, components involved in the single-molecule event, such as biological moieties, are immobilized. In certain embodiments, the components comprise biomolecules, particularly single biomolecules. The term "single biomolecule" encompasses single molecular entities, such as polypeptides, or complexes composed of multiple individual units, such as single molecular entities in which the individual units together form a functional biological moiety.

[0096] In certain embodiments, the support is a substantially planar support, i.e., it does not comprise protrusions or depressions of about 1000 nm or greater or about 100 nm or greater. In other embodiments, the support is a structured support, e.g., the support comprises depressions, such as may have a volume of about 5 x 10 -24 Increases to about 1x 10 -15 In principle, the carrier may have any design as long as a reaction space can be formed that enables a single molecule event to occur at the at least two sample sites where a single biomolecule is immobilized.

[0097] 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., from about 1.5 to about 3 in the visible range, or from about 1.5 to about 4 in the near infrared range). In other embodiments, the substrate is an optically opaque material, e.g., a metal or semimetal (such as silicon).

[0098] In certain embodiments, the substrate comprises a non-conductive material. Specific examples are glass, quartz, plastic, metal oxide-based materials, for example silicon dioxide-based materials such as glass, silicon dioxide or quartz, or composite materials comprising such materials. In other embodiments, the substrate comprises a conductive material, for example an optically transparent material such as indium tin oxide.

[0099] Typically, the thickness of the substrate is from about 10 μm to about 5 mm, particularly from about 20 μm to about 2 mm.

[0100] The surface of the carrier comprises a plurality of sample sites that are spatially separated from each other by the substrate surface. The sample sites are adapted for attachment of biomolecules. In certain embodiments, the carrier comprises a plurality of sample sites, e.g., at least 10, at least 100, at least 1,000, at least 10 4 At least 10 5 At least 10 6 and up to 10 8 10 9 Up to 10 10 or up to 10 11 A single sample point.

[0101] In some embodiments, the sample site comprises or consists of at least one electrically conductive material (e.g., a metal including a single metal or a combination of metals (e.g., an alloy or mixture of multiple different metals). For example, metals capable of attaching to a sulfur-containing moiety (e.g., in the form of a thiol or disulfide), or metals capable of attaching to a chelating moiety (e.g., a polyhistidine tag) 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 combination comprising at least two of the foregoing metals.

[0102] In certain embodiments, the sample site comprises or consists of at least one metal oxide (including a single metal oxide or a combination of metal oxides). For example, metal oxides capable of attaching to a phosphorus-containing moiety (e.g., in the form of a phosphonic acid or phosphonate), or metal oxides capable of attaching to a chelating moiety (e.g., a polyhistidine tag) are suitable. Specific examples of suitable metal oxides include TiO2 and NiO.

[0103] In further embodiments, the sample site comprises or consists of at least one non-conductive material.

[0104] The sample spots can be prepared by vapor deposition of a metal by evaporating the metal onto a support covered by a grid mask, which can be produced by electron beam lithography or an equivalent technique. The size of the holes in the grid mask can correspond to the size of the spots on the support surface. Alternatively, the spots on the support can be prepared by site-specific deposition of nanoparticles (e.g., having a size of 2-10 nm) by precise pipetting of the particles onto the support, in particular onto a support with a flat surface.

[0105] In certain embodiments, the sample spot has a size suitable for attachment of a single biomolecule.In certain embodiments, the sample spot has a diameter of about 1 nm to about 30 nm, particularly about 2 nm to about 20 nm.

[0106] In certain embodiments, the sample site is a freestanding object on the substrate surface. In certain embodiments, the sample site has a lower surface proximal to the substrate and an upper surface distal to the substrate, wherein the distance between the lower and upper surfaces defines the height of the sample site. In certain embodiments, the height is from about 50 μm to about 500 nm, particularly from about 100 μm to about 20 nm, more particularly from about 500 μm to about 10 nm, for example, about 2 nm.

[0107] The sample sites on the support can be adapted for attachment of biomolecules, for example, by covalent or non-covalent attachment. The biomolecules can be selected from polypeptides, nucleic acids, carbohydrates, and any combination thereof, such as glycosylated polypeptides or ribonucleoproteins. In certain embodiments, the biomolecule is a complex composed of several individual units (e.g., several polypeptide units, or several polypeptide and nucleic acid units).

[0108] In a specific embodiment, the biomolecule is a nucleic acid polymerase, in particular a DNA polymerase or an RNA polymerase, or a nucleic acid polymerase complex, in particular a DNA or RNA polymerase complex comprising a nucleic acid polymerase and a nucleic acid molecule. In a specific embodiment, the biomolecule is a DNA polymerase having a DNA binding cleft, in particular a family A DNA polymerase, including but not limited to Klenow, Taq or T7 DNA polymerases or any genetically modified forms thereof, or a family B polymerase, including but not limited to therminator, Phi29, RB-69 or T4 DNA polymerases or any genetically modified forms thereof. Reference is made to US 7,745,116 B2 (the contents of which are incorporated herein by reference) in this specification.

[0109] In further embodiments, the biomolecule is a nucleic acid degrading enzyme, in particular an exonuclease, or a nucleic acid degrading molecular complex, in particular a molecular complex comprising a nucleic acid degrading enzyme and a nucleic acid molecule that degrades DNA or RNA.

[0110] In yet another embodiment, the biomolecule 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).

[0111] The single molecule event to be detected occurs in a sample comprising the components required for the event (e.g., biomolecules and optionally small molecules). At least one of the components is a luminescent component comprising a luminescent group (i.e., a group capable of emitting radiation in response to being irradiated with exciting radiation). In certain embodiments, the luminescent component is a compound carrying a luminescent labeling group. In certain embodiments, the luminescent component is a compound capable of luminescence itself.

[0112] 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 capable of emitting fluorescent radiation in response to being irradiated with excitation radiation. In certain embodiments, the fluorescent component is a compound carrying a fluorescent marker group. In certain embodiments, the fluorescent component is a compound that itself is capable of fluorescing.

[0113] In certain embodiments, the sample of single molecule events comprises a plurality of different luminescent components, wherein at least some of the luminescent components have partially overlapping emission radiation spectra. For example, the sample may comprise a plurality of different fluorescent components, wherein at least some of the fluorescent components have distinguishable and partially overlapping fluorescence emission spectra.

[0114] The at least one luminescent component may be present in the sample in an immobilized form and / or in a free form. In certain embodiments, the at least one luminescent component is present in a free form.

[0115] According to the present disclosure, the excitation radiation is split into a plurality of individual radiation beams by an optical diffraction element. In certain embodiments, the excitation radiation is split into about 10 to about 1000 beams, about 50 to about 500 beams, and for example, about 100 individual radiation beams.

[0116] In certain embodiments, the optical diffraction element is a grating, such as a three-dimensional grating optionally applied to an optically transparent support.

[0117] The light passing through the optical diffraction element is split, thereby producing a predetermined diffraction pattern of a plurality of radiation beams, which can form the desired arrangement of a plurality of optical focal points in the object plane by constructive and destructive interference. The production of suitable diffractive optical elements is described in, for example, the paper (1999) of F.Nikolaef of Chalmers Institute of Technologies, the paper (2001) of M.Johansson of Chalmers Institute of Technologies, and the publication (Applied Optics 38 (1999), 1302-1310) of Johansson and Hard. Suitable materials for producing optical elements are plastics, glass and composite materials, or other materials that can be processed by photolithography and etching and have optical transparency for a given wavelength.

[0118] A single beam of excitation radiation may be directed toward a single sample or toward a group of multiple individual samples. In certain embodiments, a group of samples may contain up to about 10 6 individual samples, particularly from about 1,000 to about 10 6 or about 5,000 to about 10 5 For example, about 10,000 individual samples.

[0119] 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, for example, by using one or more highly spatially coherent lasers for generating the excitation radiation.

[0120] In certain embodiments, a plurality of excitation radiations generated by an optical diffraction element are transferred to a sample through an optically transparent substrate of a carrier on which the sample is located. In certain embodiments, the sample is located on a surface of the carrier that is remote from the surface of the carrier through which the plurality of excitation radiations enter the carrier. In those embodiments, a separate beam of excitation radiation generates a separate evanescent field on the surface of the carrier on which the sample is located. The separate evanescent field can be generated by total internal reflection. In certain embodiments, the separate evanescent field can have a diameter of about 1 nm to about 100 μm or about 10 nm to about 10 μm. In certain embodiments, the separate evanescent field can cover up to about 100,000 separate samples, in particular about 1,000 to about 100,000, or about 5,000 to about 20,000, for example about 10,000 separate samples.

[0121] In certain embodiments, multiple separate evanescent fields are generated. These separate evanescent fields may or may not overlap. In certain embodiments, the evanescent fields do not substantially overlap. In certain embodiments, the overlap between two adjacent evanescent fields is about 20% (by area) or less, or about 10% (by area) or less.

[0122] In certain embodiments, the detection device comprises a detector matrix comprising a plurality of detection pixels. The detection pixels can be optically projected onto sample points on the support surface. In certain embodiments, the detector provides an optical focus on a single sample point in order to achieve the desired accuracy when measuring single molecular events occurring at the sample point. An essential part of the present invention is the generation of a plurality of separate evanescent fields of excitation energy by splitting a laser beam using a diffractive optical element.

[0123] In certain embodiments, at least one reaction space is provided around the plurality of samples, wherein the reaction space comprises a medium (eg, a liquid medium, in particular an aqueous liquid medium) and components for the single molecule event.

[0124] In certain embodiments, the reaction space is a flow cell, thereby providing a flow of fresh components to and from a sample site. In certain embodiments, a single reaction space is provided around multiple samples. In other embodiments, a single reaction space is provided around each sample or group of samples. For example, the reaction space has a depth of about 10 nm to about 1,000 μm.

[0125] In certain embodiments, the emitted radiation path is from the sample site through the reaction space to the detection device.

[0126] In another specific embodiment, the radiation source is located above an optically transparent carrier, and the carrier comprises a plurality of sample points on its lower surface, i.e., the surface away from the radiation source. The lower surface of the carrier comprising the sample points is in contact with the reaction space (e.g., a single reaction space of a flow cell as described above). The radiation source emits excitation radiation downward in the direction of the carrier. The excitation radiation is split into a plurality of individual radiation beams, which then enter the carrier at the upper surface of the carrier, i.e., the surface close to the radiation source. Each radiation beam is reflected at the lower surface, thereby generating each evanescent field at the sample point or sample point group as described above, for example, by total internal reflection. The emission radiation path from the sample point is guided downward through the reaction space to reach the detection device.

[0127] In certain embodiments, the methods of the present disclosure include analyzing multiple subsequent single molecule events in a sample, particularly including analyzing multiple subsequent single molecule events in different samples in parallel. The term "multiple single molecule events" includes a series of consecutive single molecule events occurring at the same sample, such as a sample point on a carrier. In certain embodiments, a 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, such as up to about 10,000,000 or 100,000,000 individual single molecule events. For example, multiple single molecule events can include subsequent nucleic acid extension and / or degradation steps of single molecule nucleic acid sequence analysis.

[0128] In certain embodiments, single molecule nucleic acid sequence analysis comprises multiple steps of nucleic acid extension in which a luminescent nucleotide building block (e.g., a luminescent-labeled nucleoside polyphosphate, e.g., comprising 3 to 15 phosphate groups) is incorporated into a nucleic acid molecule in the presence of a nucleic acid polymerase (e.g., a DNA or RNA polymerase). Those embodiments may comprise detecting emitted radiation from the incorporation of a luminescent nucleotide building block (e.g., a luminescent-labeled nucleoside polyphosphate) into the nucleic acid molecule.

[0129] The present disclosure also provides an apparatus for analyzing single molecule events, comprising:

[0130] - means for providing a plurality of samples adapted for single molecule events,

[0131] an optical excitation device comprising a radiation source adapted 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,

[0132] - detection means adapted to capture emitted radiation from said plurality of samples, and

[0133] - analyzing means adapted to analyze the captured emitted radiation,

[0134] The excitation radiation path from the radiation source to the plurality of samples does not overlap with the emission radiation path from the plurality of samples to the detection device.

[0135] In certain embodiments, the device is adapted to perform the methods described above, eg, for performing single molecule nucleic acid sequence analysis.

[0136] The methods and apparatus of the present disclosure can be used to provide high throughput analysis of single molecular events. In certain embodiments, high throughput analysis includes analyzing about 10 6 to about 10 11Parallel analysis of 10 individual samples, particularly on a single support (e.g., a planar support) 6 to about 10 11 In certain embodiments, high-throughput analysis is performed in a single reaction space.

[0137] Methods and apparatus for analyzing single molecule events are disclosed, for example, in WO 2002 / 097406, WO 2003 / 052137, WO 2006 / 013110, WO 2013 / 131888, WO 2015 / 104245, WO 2017 / 001407 and WO 2018 / 104301 (the contents of which are incorporated herein by reference).

[0138] For the analysis of single molecular events, the biomolecules can be located at sample sites on the support. There, they come into contact with a sample liquid containing free reaction partners. Thus, one or more reaction spaces are defined. In particular, at least 100, at least 1,000, or at least 10,000, and up to more than 10,000, can be analyzed on a single support (e.g., a single planar support). 6 molecules.

[0139] The nucleic acid molecule whose sequence is to be determined can be selected, for example, from a DNA molecule, such as a genomic DNA fragment, a cDNA molecule, a plasmid, or the like, or from an RNA molecule, such as an mRNA molecule. The nucleic acid molecule can be derived from a genome or expression library produced from a cell or organism (e.g., a eukaryotic or prokaryotic cell or organism). This allows for the sequencing of a plurality of different nucleic acid template molecules, for example, at least 10, 100, 1,000, or 10,000, and up to 100,000, 10,000, or more. 6 or 10 7 or even more different nucleic acid molecules can be sequenced in parallel.

[0140] The nucleic acid molecule to be sequenced can be a single-stranded nucleic acid molecule in a linear or circular form (e.g., in a covalently linked circular form). In order to obtain a circular nucleic acid template, a cyclization procedure and an optional chain separation procedure can be performed on the linear nucleic acid molecule during sample preparation. Cyclization can be achieved by connection according to known protocols, for example, using DNA or RNA ligase. In some embodiments, an adapter and / or identifier molecule, i.e., a nucleic acid molecule of known sequence, can be coupled to the nucleic acid molecule.

[0141] Sequence determination may include nucleic acid extension and / or nucleic acid degradation. The sequencing process includes one or more sequencing cycles.

[0142] Nucleic acid synthetase molecules are capable of extending a primer annealed to a nucleic acid template molecule. Primer extension can be performed by the stepwise incorporation of single nucleotide building blocks at the 3' end of the growing nucleic acid chain, thereby generating a nucleic acid molecule complementary to the circular nucleic acid template sequence. The nucleic acid synthetase is selected from polymerases capable of template-specific nucleic acid polymerization, preferably DNA polymerases and RNA polymerases, such as natural or modified polymerases, including thermostable DNA polymerases.

[0143] Specific examples of suitable DNA polymerases include Taq polymerase, exonuclease-deficient Taq polymerase, E. coli DNA polymerase I, Klenow fragment, reverse transcriptase, Related polymerases (including wild-type polymerases and derivatives of such polymerases, such as exonuclease-deficient forms), T7 DNA polymerase, T5 DNA polymerase, RB69 polymerase, and the like.

[0144] Nucleic acid degrading enzyme molecule can progressively cut out single nucleotide building blocks from nucleic acid molecules.Preferably use exonuclease, more preferably use the single-stranded exonuclease of 3' → 5' direction or 5' → 3' direction degradation.The exonuclease that particularly preferably uses is 3' → 5' exonuclease, such as Escherichia coli exonuclease I and Escherichia coli exonuclease III, and 5' → 3' exonuclease, such as T7 exonuclease, Escherichia coli exonuclease II and Escherichia coli exonuclease VIII.In addition, can use the exonuclease activity of various polymerases for example Klenow fragment, Taq polymerase or T4 polymerase.

[0145] Nucleic acid synthetase molecule and linear or circular nucleic acid template molecule (for example single-stranded DNA or RNA molecule) and with nucleic acid template molecule annealing or can be contacted with its annealed primer molecule.Primer molecule is preferably single-stranded nucleic acid or nucleic acid analog molecule with free 3 ' end, and it can extend by the enzymatic reaction catalyzed by fixed nucleic acid synthetase molecule.Select the length of primer molecule, to allow under reaction conditions and template effective annealing.Usually, the length of primer molecule is at least 8, at least 10, at least 12 or at least 15 nucleotide, for example up to 20, 25, 50 or 100 nucleotide, or even more nucleotide.In some embodiments, primer for example resists the digestion of nucleic acid degrading enzyme molecule by mixing the linkage between nucleotide analog building block and / or the nucleotide building block, and it is stable to degraded.In other embodiments, primer is sensitive to the digestion of nucleic acid degrading enzyme molecule.

[0146] The primer sequence 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 an unknown nucleic acid sequence. In other embodiments, the primer may be able to anneal to a known sequence portion of the nucleic acid template molecule. In this embodiment, known adapters and / or identifier sequences can be incorporated into the nucleic acid template molecule. The primer can be unlabeled or contain a fluorescent marker group.

[0147] Furthermore, the presence of luminescent nucleotides is required, for example, nucleotide building blocks carrying at least one fluorescent marker group. Preferably, each different nucleotide building block (A, G, C, T / U) comprises a different fluorescent marker group.

[0148] The fluorescent labeling group can be selected from known fluorescent labeling groups for labeling biopolymers (especially nucleic acids), such as fluorescein dyes, rhodamine, oxazines such as Evoblue or Gnothis Blue, phycoerythrin, Cy3, Cy5, IR dyes or their derivatives.

[0149] The nucleotide building block may carry (i) a fluorescent marker group that is retained in the building block when the building block is incorporated into a nucleic acid molecule during primer extension catalyzed by a nucleic acid synthetase molecule, and / or (ii) a fluorescent marker 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 synthetase molecule. The fluorescent marker group that is retained in the building block is preferably attached to an α-phosphate group, a sugar, and / or a nucleobase.

[0150] In a specific embodiment, the fluorescent marker group retained in the building block is, for example, connected to the core base by a linker (optionally comprising heteroatoms, such as N, O or S atoms) having a chain length of up to 15, preferably 10-12 carbon atoms. The fluorescent marker group that is excised when the building block is incorporated into the nucleic acid molecule can be connected to, for example, the terminal phosphate group of a polyphosphate building block (including but not limited to hexaphosphate, pentaphosphate, tetraphosphate or triphosphate building blocks), such as the gamma-phosphate group of a triphosphate building block. In certain embodiments, a building block comprising both the fluorescent marker group remaining after (i) incorporation and the fluorescent marker group excised during (ii) incorporation is selected. In this case, a fluorescent group that can, for example, interact by quenching and / or energy transfer can be selected.

[0151] In the case of direct sequencing of nucleic acid molecules using nucleic acid degrading enzyme molecules, the nucleic acid molecules to be sequenced will contain a fluorescent marker group. On the other hand, if the nucleic acid molecules are used as templates in primer extension, the nucleic acid molecules to be sequenced may not contain a fluorescent marker group.

[0152] The sequencing procedure can include a step of generating a nucleic acid molecule that has been incorporated during primer extension catalyzed by a nucleic acid synthetase molecule and / or a second step of cleaving individual nucleotide building blocks from the generated nucleic acid molecule, catalyzed by a nucleic acid degrading enzyme molecule. Depending on the type of fluorescent label, nucleic acid sequence determination can be performed during the primer extension and / or degradation process.

[0153] Sequence determination in the primer extension process relates to the use of a nucleotide building block that carries a fluorescent marker group, and when the building block is incorporated into the nucleic acid molecule, the fluorescent marker group is cut off from the building block. In this case, the time-dependent fluorescence change caused by the excision of the fluorescent marker group from the nucleotide building block can be measured. Sequence determination in the nucleic acid degradation process relates to the use of a nucleotide building block that carries a fluorescent marker group, and when the building block is incorporated into the nucleic acid molecule, the fluorescent marker group remains in the building block. When the marked nucleotide building block is released from the nucleic acid molecule, the progressive cutting of the single nucleotide building block from the nucleic acid molecule causes a time-dependent change in fluorescence. In certain embodiments, sequence determination can also be carried out during extension and degradation, that is, when using a nucleotide building block, the nucleotide building block both carries the fluorescent marker group that remains in the building block and also carries the fluorescent marker group that is cut off from the building block when the building block is incorporated into the nucleic acid molecule. In this embodiment, two fluorescent groups can be identical or different.

[0154] In some embodiments, the method comprises one or more cycles of nucleic acid synthesis and nucleic acid degradation to determine the base sequence of the nucleic acid template. Nucleic acid synthesis comprises extension of a primer annealed to the nucleic acid template molecule catalyzed by a nucleic acid synthetase molecule, wherein a nucleic acid molecule complementary to the sequence of the nucleic acid template is produced. In a next step, the produced nucleic acid molecule is degraded by a nucleic acid degrading enzyme molecule.

[0155] When the Nucleotide building block is incorporated into the nucleic acid molecules of elongation, fluorescent time-dependent variation can take place, and this can be detected as described above.Preferably, the Nucleotide building block is incorporated into the nucleic acid molecules of extension and is relevant to the detectable increase of fluorescence, preferably relevant to the transient increase of fluorescence.For example, the Nucleotide building block can be used, and described Nucleotide building block carries a fluorescent marker group on a part for molecule (for example, on gamma-phosphate group), and when building block is incorporated into primer, described fluorescent marker group is excised.

[0156] When a nucleotide building block is removed from a synthetic nucleic acid molecule, a time-dependent change in fluorescence can be measured due to the interaction of the fluorescent marker group incorporated into the nucleic acid chain with adjacent groups, for example, chemical groups of the nucleic acid (particularly nucleobases, such as G) or / and adjacent fluorescent marker groups, and due to quenching processes and / or energy transfer processes, these interactions result in a change in fluorescence, in particular a change in fluorescence intensity, compared to the fluorescent marker group in "isolated" form. The removal of a single nucleotide building block by cleavage changes the overall fluorescence, for example, the fluorescence intensity of the immobilized nucleic acid chain, and this change is a function of the removal of the single nucleotide building block by cleavage, i.e., a function of time.

[0157] In certain embodiments, the association of labeled nucleotides with biomolecule complexes is detected by measuring the polarization of emitted photons. The polarization of excited-state photons is altered by the rotational motion of the luminescent nucleotide labels and can be used to identify freely mobile, reverse-bound labeled nucleotides during polymerization.

[0158] During the extension and / or degradation process, such time-dependent changes in fluorescence of multiple nucleic acid molecules can be recorded in parallel and correlated with the base sequence of a single nucleic acid strand. Preferably, fluorescent marker groups are used which, when incorporated into a nucleic acid strand, are at least partially quenched, such that the fluorescence intensity increases after the nucleotide building block containing the marker group or the adjacent building block causing the quenching is removed by cleavage.

[0159] During the incorporation and / or removal of a single nucleotide building block, changes in the fluorescence intensity of the nucleic acid chain and / or the incorporated or removed nucleotide building block can be measured due to quenching or energy transfer processes. This temporal change in fluorescence intensity depends on the base sequence of the nucleic acid chain being studied and can therefore be correlated with the sequence.

[0160] The complete sequence of a nucleic acid molecule can be determined by using a mixture of nucleotide building blocks that are labeled at all four different bases, for example at A, G, C and T, or at a combination of two or three different bases. Where appropriate, a "sequence identifier" (i.e., a labeled nucleic acid of known sequence) can also be attached to the nucleic acid strand to be investigated, for example by an enzymatic reaction using a ligase and / or a terminal transferase, so that a known fluorescence pattern is initially obtained at the start of sequencing and only thereafter a fluorescence pattern corresponding to the unknown sequence to be investigated is obtained.

[0161] Detection includes irradiating the excitation radiation from the radiation source into the carrier, preferably by laser or another suitable light source, to cause the excitation of the fluorescent marker group. In certain embodiments, the radiation source includes a plurality of different lasers emitting radiation at different wavelengths. In this regard, one or more laser beams can be used, such as an expanded laser beam (with a cross section of about 1-20 mm), and / or a plurality of laser beams. Detection preferably includes multi-point fluorescence excitation by laser, such as a lattice of laser points produced by a diffraction optical element (see WO 2002 / 097406) or a quantum well laser.

[0162] Fluorescence emission of multiple nucleic acid chains can be detected in parallel using a detection device (e.g., a detector matrix), including, for example, an electronic detector matrix (e.g., a CCD camera), a CMOS detector matrix (e.g., a CMOS camera), or an avalanche photodiode matrix. Detection can be performed in such a way that fluorescence excitation and detection are performed in parallel on some or all of the nucleic acid chains being studied. Preferably, fluorescence emitted substantially perpendicular to the surface of the carrier is detected, the fluorescence passing through the reaction space or through the carrier.

[0163] For example, detection can be performed by single molecule detection, for example by fluorescence correlation spectroscopy, which involves the transfer of very small, preferably confocal, volume elements (e.g. 10 -21 to 10 -10 1) exposure to excitation light from a laser or another suitable light source, which excites a receptor present in the measurement volume, causing the latter to emit fluorescence, the fluorescence emitted from the measurement volume being measured by a photodetector, and the variation of the measured emission over time being correlated with the concentration of the analyte, making it possible to identify single molecules in the measurement volume at appropriately high dilutions. Details of the procedures and instruments 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 also 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.

[0164] Alternatively or additionally, detection can also be performed by means of time-resolved decay measurements (called "time gating", as described, for example, by Rigler et al., "Picosecond Single Photon Fluorescence Spectroscopy of Nucleic Acids" in "Ultrafast Phenomena", D.H. Auston, ed., Springer 1984 (the content of which is incorporated herein by reference). Here, fluorescent molecules are excited in the measurement volume and the detection interval is subsequently opened on the photodetector, for example at time intervals of ≥100 ps. In this way, the background signal generated by the Raman effect can be kept sufficiently low to enable the detection of single molecules in a substantially interference-free manner.

[0165] The methods and apparatus of the present disclosure are also applicable to the analysis of other single molecule events for which there is a high demand for high-throughput single molecule analysis (ie, a single biomolecule bound to a selected spot for analysis).

[0166] In certain embodiments, the present disclosure relates to single molecule analysis of receptor-ligand interactions, eg, involving binding of a receptor protein to a sample site and subsequent study of its interaction with its ligand, eg, in drug development.

[0167] In other embodiments, the present disclosure relates to single molecule analysis of hybridization events, for example, comprising the attachment of short single-stranded nucleic acid molecules (e.g., DNA or RNA molecules), for example, ranging in length from 3 to 300 nucleotides, followed by the addition of a sample containing a complementary nucleic acid molecule and observation of any hybridization events. Application areas may be, for example, the detection of viral RNA / DNA, the detection of bacterial DNA / RNA, and the detection of short segments of DNA from cancer cells in the bloodstream.

[0168] Furthermore, the present disclosure is explained in detail by referring to the following specific embodiments.

[0169] Figure 1 An embodiment of the present disclosure is shown. A laser beam (12) is split into a plurality of individual beams, for example four individual beams (16), by a diffractive optical element (14). The individual beams (14) are directed to a sample point (18) located on a carrier (20), wherein the individual beams (14) produce a predetermined optical focus pattern. Light (22) emitted from the focus at the sample point (18) is directed by an optical device (24) to a detector (26). The difference between this embodiment and the prior art embodiment (previous slide) is that the radiation source light path and the emission light path are different throughout the entire light path and do not overlap.

[0170] Figure 2Further embodiments of the present disclosure are shown wherein the detection is based on the evanescent field. Figure 2 In , a single evanescent field is depicted, for example 1 out of a total of 10,000 evanescent fields.

[0171] An incident single light beam (30) generated by passing the excitation light through an optical diffraction element (not shown) passes through a gaseous medium (32) to an optically transparent carrier (34) made, for example, of silicon dioxide. It enters the carrier (34) at a surface (34a) away from a surface (34b) where a sample point (36) is located. Binding molecules (38) are attached to the surface of the sample point (36) and mediate the fixation of a single biomolecule or a single molecule complex (40) (for example, DNA or RNA polymerase, or a complex comprising DNA or RNA polymerase and a nucleic acid template and a primer). The sample point (36) is surrounded by a reaction space (42) that contains a medium (for example, an aqueous medium) and components required for a single molecule event, such as a fluorescently labeled nucleotide component. The incident light beam (30) is reflected at the surface (34b). As a result, totally reflected light (44) and an evanescent field (46) are generated. The evanescent field (46) comprises a single sample point (36) or - as shown here - a group of sample points (36), for example up to 100,000 sample points, typically about 10,000 sample points, and can have a diameter of, for example, 1 nm to 100 μm. Because the excitation light is split into multiple individual beams, multiple evanescent fields can be generated on the carrier surface (34b). Emission light from single molecule events occurring at the sample points (36) is transmitted to a detector (not shown).

[0172] Figure 3 Another embodiment of the present disclosure is shown. A carrier (50) is provided having a sample point (52) on its surface on which a single molecule (not shown) is immobilized. The sample point (52) is in contact with a single reaction space (54), which is a flow cell comprising an inlet (56) for the inflow of a medium containing fresh components for interacting with the single molecule and an outlet (58) for the outflow of a medium containing used components. Emitted light (not shown) from the sample point (52) passes through the reaction space (54) to a detector (60). The detector comprises pixels that are optically projected onto the sample point (52) on the surface of the carrier (50).

Claims

1. A method for analyzing single molecule events, comprising: (a) providing a plurality of samples in which a single molecular event occurs, wherein the plurality of samples are present on a carrier, wherein the carrier comprises an optically transparent substrate and a plurality of sample spots on a surface of the carrier, wherein the sample spots are spatially separated from one another, and wherein the samples are present on the sample spots, and wherein a component of the single molecule event is immobilized to at least one sample site, and wherein the immobilized component comprises a single biomolecule, (b) irradiating the plurality of samples with an optical excitation device comprising a radiation source, 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 to the plurality of samples, (c) capturing the emitted radiation from the plurality of samples by a detection device, and (d) analyzing the captured emitted radiation, wherein an excitation radiation path from the radiation source to the plurality of samples does not overlap with an emission radiation path from the plurality of samples to the detection device, wherein the plurality of light beams generated by the optical diffraction element are transferred to the plurality of samples through an optically transparent base of the carrier, wherein the plurality of samples are located on a surface of the carrier remote from a carrier surface through which the excitation radiation enters the carrier, wherein the individual beams of excitation radiation generate individual evanescent fields on the carrier surface where the plurality of samples are located by total internal reflection, and Each individual evanescent field illuminates a set of sample points.

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

3. The method of any one of the preceding claims, wherein the individual beams of excitation radiation are directed toward a group comprising a plurality of individual samples, wherein the group comprises up to about 10 6 individual samples, particularly from about 1,000 to about 10 6 or about 5,000 to about 10 5 For example, about 10,000 individual samples.

4. The method of any of the preceding claims, wherein 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 cover up to about 10 6 individual samples, particularly from about 1,000 to about 10 6 or about 5,000 to about 10 5 For example, about 10,000 individual samples.

5. The method according to any of the preceding claims, wherein the detection device comprises a detector matrix comprising a plurality of detection pixels, wherein the detection pixels are optically projected onto sample spots on the carrier surface.

6. The method of any of the preceding claims, wherein at least one reaction space is provided around the plurality of samples, wherein the reaction space comprises a medium and components for the single molecule events. The method of claim 6 , wherein the emitted radiation path passes through the reaction space.

8. The method of any of the preceding claims, wherein the single molecule event comprises sequence analysis of a single nucleic acid molecule.

9. An apparatus for analyzing single molecule events, comprising: - means for providing a plurality of samples adapted for a single molecule event, wherein the plurality of samples are present on a carrier, wherein the carrier comprises an optically transparent substrate and a plurality of sample spots on a surface of the carrier, wherein the sample spots are spatially separated from each other, and wherein the samples are present on the sample spots, and wherein a component of the single molecule event is immobilized to at least one sample site, and wherein the immobilized component comprises a single biomolecule, an optical excitation device comprising a radiation source adapted 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 - analyzing means adapted to analyze said captured emitted radiation, wherein an excitation radiation path from the radiation source to the plurality of samples does not overlap with an emission radiation path from the plurality of samples to the detection device, wherein the apparatus is adapted to pass the plurality of light beams generated by the optical diffraction element through an optically transparent substrate of a carrier to the plurality of samples, wherein the plurality of samples are located on a surface of the carrier remote from a carrier surface through which the excitation radiation enters the carrier. wherein the individual beams of excitation radiation generate individual evanescent fields on the carrier surface where the plurality of samples are located by total internal reflection, and Each individual evanescent field illuminates a set of sample points.

10. The apparatus of claim 9 adapted to perform single molecule nucleic acid sequence analysis.

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

12. The use of claim 11, wherein the high-throughput analysis comprises analyzing approximately 10 6 to about 10 11 Parallel analysis of individual sample points.

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