Protein-resistant surfaces coated with selected areas

Abstract

The present disclosure relates to a support, optionally comprising a transparent substrate and at least one sample spot on a surface of the support, a device comprising the support for determining single molecular events, and a method in which the support is used for determining single molecular events.

Description

[Technical Field]

[0001] explanation The present disclosure relates to a support comprising an optically transparent substrate and at least one sample spot on a surface of the support, a device for analyzing single molecular events comprising the support, and a method for analyzing single molecular events in which the support is used. [Background technology]

[0002] background The use of protein-resistant surfaces is essential for the development of biomedical devices and systems, from biosensors to drug delivery systems. This is also essential for single-molecule studies of biomolecular interactions, where any signal from nonspecifically bound proteins on the surface of the test device would interfere with the measurement.

[0003] One of the most commonly used passivation techniques is the use of protein-repellent coatings, in which polyethylene glycol (PEG) molecules are chemically linked to the underlying substrate and arranged in a thin, brush-like layer in many configurations. [1] However, PEG passivation is often deficient, and many techniques have been developed to improve its coverage and effectiveness. [2-4] Other known organic passivation films have similar deficiencies. [4]

[0004] Inorganic films of diamond-like carbon (DLC), or amorphous carbon, are highly resistant to protein adsorption. Carbon films are highly uniform and extremely robust, with mechanical strength far exceeding that of organic films. They are relatively unaffected by environmental and ambient conditions (e.g., they do not need to be stored in water), their properties make them suitable for reuse, and they have been found to resist protein adhesion, making them excellent candidates for coatings on biomedical devices such as implants [5, 6]. DLC films have excellent mechanical and tribological properties [7, 8] and low surface energy. They have found use as wear-resistant coatings in a wide variety of applications [9].

[0005] Further reduction in surface energy, and therefore wettability, can be achieved by incorporating fluorine into the film. Fluorine can be introduced either during DLC ​​deposition by chemical vapor deposition

[10] or by exposure to fluorine-containing plasmas, such as from fluorocarbon gases (e.g., C4F8, CHF3, NF3, or SF6). Fluorine atoms reside at or within interatomic distances of the surface

[10] . The result is a highly hydrophobic (or superhydrophobic) surface [11, 12] that has been demonstrated to be resistant to the adhesion of many species, and particularly to the adhesion of cells and biomolecules [6, 13, 14].

[0006] However, the inventors are unaware of the use of optically transparent surfaces coated with inorganic carbon layers in single molecule studies of biomolecular interactions involving optical detection in a reaction space located above the coated support. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention Carbon film coated substrate In a first aspect, the present disclosure relates to a substrate area-selectively coated with a carbon film, and devices and methods in which the substrate is used for analyzing events, e.g., single molecular events, associated with the emission of electromagnetic radiation.

[0008] According to this aspect, the support includes a substrate having a surface selectively coated with a diamond-like carbon (DLC) and / or amorphous carbon film, rendering it resistant to adhesion of proteins and other biological moieties in the coated areas while allowing selective binding of the biological moieties to uncoated areas. The carbon film can be deposited on the surface of the substrate by any suitable method, including, but not limited to, physical vapor deposition, chemical vapor deposition, and / or atomic layer deposition. Its anti-adhesion properties can be further enhanced by fluorination, for example, by treatment with a fluorine-based plasma. The carbon film is mechanically robust and chemically stable, and it can withstand multiple uses with minimal cleaning and / or reactivation steps. Thus, carbon-coated surfaces can replace surfaces treated with conventional protein-repellent agents such as polyethylene glycol (PEG).

[0009] In the first embodiment of this aspect, the present disclosure relates to a support, the support comprises substrate and at least one sample spot on the surface of the support, the substrate is at least partially coated with diamond-like carbon and / or amorphous carbon layer, and the layer of diamond-like carbon and / or amorphous carbon does not extend over at least one binding spot.In certain embodiments, the substrate is optionally a transparent substrate.

[0010] A further embodiment relates to the use of the support described above for analyzing an event at at least one sample spot, wherein the event is associated with the emission of electromagnetic radiation. In a particular embodiment, the event is a single molecule event.

[0011] In certain embodiments, the carbon layer is a layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon.

[0012] In a further embodiment, the support can be reused after being subjected to a cleaning procedure, in which the biological moieties attached to at least one sample spot are removed.The cleaning efficiency of the used support can be improved when the carbon layer is a layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon.

[0013] Further embodiments include: (i) providing a substrate containing at least one sample spot on its surface; and (ii) coating the surface of the substrate with a layer of diamond-like and / or amorphous carbon; Including, The present invention relates to a method for producing such a support, wherein the material of the sample spots is selected so as to be insensitive to the coating in step (iii), so that no layer of diamond-like and / or amorphous carbon extends over at least one sample spot.

[0014] Further embodiments include: (i) coating the surface of a substrate with a layer of diamond-like and / or amorphous carbon; and (ii) depositing at least one sample spot on the surface of said layer; The present invention relates to a method for producing the support, comprising:

[0015] Further embodiments include: (i) coating the surface of a substrate with a layer of diamond-like and / or amorphous carbon; (ii) removing the layer in selected areas of the surface; and (iii) depositing at least one sample spot in the selected area from which the layer was removed. The present invention relates to a method for producing the support, comprising:

[0016] Further embodiments include: (i) providing the support; (ii) immobilizing a biological moiety, in particular a biomolecule, on at least one sample spot of the support; and (i) analyzing events associated with said biological moieties, in particular events associated with said biomolecules, by detecting electromagnetic radiation from said sample spots; The present invention relates to a method for analyzing an event, including:

[0017] In certain embodiments, the event is a single molecular event and the biomolecule is a single biomolecule.

[0018] Further embodiments include: (i) a support adapted to immobilize a biological moiety, in particular a biomolecule, on at least one sample spot of said support; (ii) a means for directing radiation to at least one sample spot on the support; and (iii) means for analyzing events on said at least one sample spot by detecting electromagnetic radiation from said spot; The present invention relates to a device for analyzing events, including

[0019] In certain embodiments, the event is a single molecular event and the biomolecule is a single biomolecule.

[0020] Cleaning previously used supports A second aspect described herein is a method for cleaning a previously used substrate suitable for devices and methods for analyzing events, e.g., single molecular events, where the events are associated with the emission of electromagnetic radiation. In accordance with this aspect, the substrate preferably comprises a carbon layer that is a layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon.

[0021] In certain embodiments, the cleaning procedure involves subjecting the used support to (a) treatment with an alkaline solution, (b) treatment with an acidic / oxidizing solution, optionally (c) exposure to O2 plasma, and (d) optionally exposure to a fluorine-containing plasma.

[0022] Unless otherwise stated, features of the first aspect also apply to the second aspect.

[0023] An embodiment of the second aspect comprises: (i) Providing a support comprising a substrate and at least one sample spot on a surface of the support, wherein a biological moiety is attached to the at least one sample spot. (ii) subjecting the support from step (i) to treatment with an alkaline solution, wherein said treatment is carried out at an elevated temperature; (iii) rinsing the support after step (ii) if necessary; (iv) subjecting the support after step (ii) or (iii) to treatment with an acidic / oxidizing solution comprising a strong inorganic acid and a peroxide; (v) rinsing the support after step (iv) if necessary; (vi) optionally drying the support after step (iv) or (v) with an inert gas; (vii) optionally subjecting the support after step (iv), (v) or (vi) to a plasma treatment, for example treatment with O2 plasma and / or treatment with a fluorine-containing plasma. The present invention relates to a method for cleaning a previously used substrate, comprising:

[0024] In certain embodiments, the substrate is an optically transparent substrate.

[0025] In a further embodiment of this aspect, biological moieties such as biomolecules are attached to the cleaned support after step (vii).Furthermore, the support surface surrounding the sample spot can be passivated, i.e., treated with a protein adhesion repellent, to inhibit the adhesion of biological moieties such as biomolecules and / or other sample components.The support is then ready to be used for a new analysis of events, for example, single molecular events.

[0026] Item In the following, certain items of specification are described herein: 1. A support comprising a substrate and at least one sample spot on a surface of the support; A support, wherein the substrate is at least partially coated with a layer of diamond-like carbon and / or amorphous carbon, and wherein the layer of diamond-like carbon and / or amorphous carbon does not extend over at least one sample spot.

[0027] 2. The support of item 1, which is at least substantially planar.

[0028] 3. Structured, support for items 1 or 2.

[0029] 4. The support of any one of the preceding items, wherein the substrate is optically transparent.

[0030] 5. The support of any one of the preceding items, wherein the substrate comprises a material having a refractive index of at least 1.01.

[0031] 6. The support of any one of the preceding items, wherein the substrate comprises a non-conductive material.

[0032] 7. The support of any one of the preceding items, wherein the substrate comprises a material selected from the group consisting of silica, quartz, and glass.

[0033] 8. The support of any one of the preceding items, wherein the substrate has a thickness of about 10 μm to about 5 mm, in particular about 20 μm to about 2 mm.

[0034] 9. The support of any one of the preceding items, comprising a plurality of sample spots, e.g., at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 or more sample spots.

[0035] 10. The support of any one of the preceding items, wherein at least one sample spot comprises at least one electrically conductive material, e.g., a metal, including a pure metal or a combination of metals, e.g., an alloy or mixture of multiple different metals.

[0036] 11. The support of item 10, wherein at least one metal is capable of forming a bond with sulfur, for example in the form of a thiol or disulfide.

[0037] 12. The support of any one of items 10 to 11, wherein at least the metal has a positive electrochemical potential.

[0038] 13. The support according to any one of items 10 to 12, wherein the at least one metal is selected from Au, Cu, Ni, Pt, Pd, Rh, Ir, Os, Ru and any combination comprising at least two of said metals.

[0039] 14. The support of any one of items 1 to 9, wherein at least one sample spot comprises at least one metal oxide, such as TiO2, NiO, or ITO.

[0040] 15. The support of any one of the preceding items, wherein at least one sample spot has a diameter of about 1 nm to about 100 μm, in particular a diameter of about 2 nm to about 50 μm.

[0041] 16. The support of any one of the preceding items, wherein at least one sample spot has a diameter of about 1 nm to about 100 nm, particularly a diameter of about 2 nm to about 50 nm, and more particularly a diameter of about 5 nm to about 20 nm.

[0042] 17. The support of any one of the preceding items, wherein at least one sample spot has an upper surface distal to the substrate and the upper surface is flush with the surrounding layer of diamond-like carbon and / or amorphous carbon.

[0043] 18. The support of any one of items 1 to 17, wherein at least one sample spot has an upper surface distal to the substrate, and wherein the upper surface of the sample spot is above a surrounding layer of diamond-like carbon and / or amorphous carbon.

[0044] 19. The support of any one of items 1 to 17, wherein at least one sample spot has an upper surface distal to the substrate, and wherein the upper surface of the sample spot is below a surrounding layer of diamond-like carbon and / or amorphous carbon.

[0045] 20. The support of any one of the preceding items, wherein at least one sample spot has a lower surface proximal to the substrate and an upper surface distal to the substrate, the distance between the lower surface and the upper surface defining the height of the sample spot, and 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, e.g., about 2 nm.

[0046] 21. The support of any one of the preceding items, wherein at least one sample spot extends through the carbon layer so that its underside is in direct contact with the surface of the substrate.

[0047] 22. The support of any one of items 1 to 20, wherein at least one sample spot does not extend through the carbon layer, such that its underside is in direct contact with the surface of the carbon layer.

[0048] 23. The support of any one of items 1 to 20, wherein at least one sample spot is located on a pillar etched into a planar substrate, and wherein a layer of diamond-like carbon and / or amorphous carbon coats the sidewalls of the pillar and the surface of the planar substrate.

[0049] 24. The support of any one of the preceding items, wherein the layer of diamond-like carbon and / or amorphous carbon has a thickness of from about 0.3 nm to about 200 μm, in particular from about 3 nm to about 9 nm, from about 5 nm to about 100 μm, from about 10 nm to about 100 μm, or from about 1 μm to about 50 μm.

[0050] 25. The support of any one of the preceding items, wherein the layer of diamond-like carbon and / or amorphous carbon is fluorinated diamond-like carbon and / or fluorinated amorphous carbon.

[0051] 26. The support of any one of the preceding items, wherein at least one sample spot is adapted for attachment, e.g., covalent or non-covalent attachment, of a biological moiety selected from a biomolecule, a cell fraction, an organelle, or a cell.

[0052] 27. The support of any one of the preceding items, wherein at least one sample spot is adapted for attachment, e.g., for covalent or non-covalent attachment, of a single biomolecule.

[0053] 28. The support of any one of the preceding items, wherein a biological moiety selected from a biomolecule, a cell fraction, an organelle, or a cell is attached to at least one of the sample spots.

[0054] 29. The support of any one of the preceding items, wherein a single biomolecule is attached to at least one of the sample spots.

[0055] 30. The support according to any one of items 26 to 29, wherein the biomolecule is selected from polypeptides, nucleic acids, carbohydrates, and any combination thereof, and the biomolecule is in particular a nucleic acid-polymerizing enzyme, such as an RNA polymerase or a DNA polymerase, or a nucleic acid-degrading enzyme, such as an exonuclease.

[0056] 31. The support of any one of the preceding items, which is a previously used and cleaned support that can be obtained by subjecting the used support to (a) treatment with an alkaline solution, (b) treatment with an acidic / oxidizing solution, optionally (c) exposure to O2 plasma, and optionally (d) exposure to a fluorine-containing plasma.

[0057] 32. Use of a support according to any one of items 1 to 31 for analyzing events occurring on at least one sample spot, wherein said events are associated with the emission of electromagnetic radiation.

[0058] 33. Use of the support of any one of items 1 to 31 for analyzing a single molecular event occurring on at least one sample spot, wherein said single molecular event is associated with the emission of electromagnetic radiation.

[0059] 34. Use of the support according to any one of items 1 to 31 for separately analyzing a plurality of events, in particular single molecular events each occurring on at least one sample spot, wherein the single molecular events are associated with the emission of electromagnetic radiation.

[0060] 35. Use of item 34, in which multiple events, especially single molecular events, are analyzed in parallel.

[0061] 36. The use of any one of items 32 to 35, wherein the single molecular event comprises the determination of a nucleic acid sequence.

[0062] 37. Nucleic acid sequence determination - providing at the sample spot (i) a single nucleic acid molecule, (ii) a nucleic acid-synthesizing enzyme molecule and / or a nuclease molecule, and (iii) a fluorescently labeled nucleotide building block in free form and / or incorporated into the nucleic acid molecule; - performing an enzymatic reaction in which nucleotide building blocks are incorporated into and / or cleaved from said single nucleic acid molecule; and - individually determining the base sequence of a nucleic acid molecule based on time-dependent fluorescence changes that occur as nucleotide building blocks are incorporated into and / or cleaved from said single nucleic acid molecule. Use of item 36, including:

[0063] 38. Use of item 37, wherein nucleic acid-synthesizing enzyme molecules and / or nucleic acid-degrading enzyme molecules are immobilized on the sample spots.

[0064] 39. Use of any one of items 32 to 38, wherein the support has previously been used for the analysis of an event, e.g., a single molecular event, and has subsequently been subjected to a cleaning procedure in which biological moieties, such as biomolecules, attached to at least one sample spot have been removed.

[0065] 40. A method for producing a support according to any one of items 1 to 31, comprising: (i) providing a substrate containing at least one sample spot on its surface; and (ii) coating the surface of the substrate with a layer of diamond-like carbon and / or amorphous carbon; wherein the material of the sample spots is selected to be insensitive to coating in step (iii), whereby a layer of diamond-like and / or amorphous carbon does not extend over at least one sample spot.

[0066] 41. A method for producing a support according to any one of items 1 to 31, comprising: (i) coating the surface of a substrate with a layer of diamond-like carbon and / or amorphous carbon; and (ii) depositing at least one sample spot on the surface of said layer; A method comprising:

[0067] 42. A method for producing a support according to any one of items 1 to 31, comprising: (i) coating the surface of a substrate with a layer of diamond-like carbon and / or amorphous carbon; (ii) removing the layer in selected areas of the surface; and (iii) depositing at least one sample spot in the selected area from which the layer was removed. A method comprising:

[0068] 43. A method for analyzing an event, e.g., a single molecular event, comprising: (ii) providing a support according to any one of items 1 to 31; (iii) immobilizing a biological moiety, in particular a single biomolecule, on at least one sample spot of the support; and (iv) analyzing events associated with the biological moiety, in particular single molecular events associated with the single biomolecule, by detecting electromagnetic radiation from the sample spot. A method comprising:

[0069] 44. The method of item 43, wherein the single molecular event comprises sequence analysis of a single nucleic acid molecule.

[0070] 45. A device for analyzing single molecular events, comprising: (i) a support according to any one of items 1 to 31; (ii) a means for directing radiation to at least one sample spot on the support; and (iii) means for analyzing single molecular events on said at least one sample spot by detecting electromagnetic radiation from said sample spot; Including, the device.

[0071] 46. ​​The device of item 45 adapted for sequence analysis of a single nucleic acid molecule.

[0072] 47. A method of cleaning a previously used support comprising subjecting the used support to (a) treatment with an alkaline solution, (b) treatment with an acidic / oxidizing solution, optionally (c) exposure to an O2 plasma, and optionally (d) exposure to a fluorine-containing plasma.

[0073] 48. Methods of item 47, including: (i) Providing at least one sample spot on the surface of the substrate and support, wherein a biological moiety is attached to the at least one sample spot. (ii) subjecting the support from step (i) to treatment with an alkaline solution containing an alkaline phosphate, e.g., potassium phosphate, and optionally a surfactant and / or a chelating agent, wherein the treatment is carried out at elevated temperature, in particular at boiling; (iii) optionally rinsing the support after step (ii), e.g., with water; (iv) subjecting the support after step (ii) or (iii) to treatment with an acidic / oxidizing solution comprising a strong inorganic acid, e.g., sulfuric acid, and a peroxide, e.g., hydrogen peroxide; (v) optionally rinsing the support after step (iv), e.g., with water and / or an anhydrous organic solvent, e.g., ethanol; (vi) optionally drying the support after step (iv) or (v) with an inert gas, such as Ar, N2, or any mixture thereof; (vii) subjecting the support after step (iv), (v) or (vi) to a plasma treatment, in particular an O2 plasma treatment and / or a fluorine-containing plasma treatment.

[0074] 49. The method of items 47 or 48, wherein the support has previously been used to analyze an event, e.g., a single molecular event, wherein the event is associated with the emission of electromagnetic radiation.

[0075] 50. The method of any one of items 47 to 49, wherein the support in step (i) comprises a substrate and at least one sample spot on a surface of the support, wherein the substrate is at least partially coated with a layer of diamond-like carbon and / or amorphous carbon, and wherein the diamond-like carbon and / or amorphous carbon layer does not extend over the at least one sample spot.

[0076] 51. The method of any one of items 47 to 50, wherein the support in step (i) comprises a substrate and at least one sample spot on a surface of the support, wherein the substrate is at least partially coated with a layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon, and wherein the layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon does not extend over the at least one sample spot.

[0077] 52. The method of any one of items 47 to 51, wherein the support comprises a substrate and at least one sample spot on a surface of the support, wherein the substrate is at least partially coated with a layer of organic passivating material, and wherein the layer of organic passivating material does not extend over the at least one sample spot.

[0078] 53. The method of any one of items 47 to 52, comprising attaching a biological moiety, such as a biomolecule, to the cleaned support after step (vii).

[0079] 54. The method of item 53, optionally including passivating the substrate and attaching a biomolecule to at least one sample spot, wherein the support is ready for use in a de novo analysis of an event, e.g., a single molecular event.

[0080] 55. (viii) activating the surface of at least one sample spot, for example, subjecting the support after step (vii) to treatment with a thiol reagent; (ix) optionally rinsing the support after step (viii) with an organic solvent, such as absolute ethanol; (x) optionally drying the support after step (viii) or (ix) with an inert gas, such as Ar, N2, or any mixture thereof; (xi) optionally regenerating the surface of the support, e.g., passivating the substrate by treatment with a PEGylation reagent such as an alkoxy PEG silane, e.g., a methoxy PEG silane, or by coating the surface of the support with a layer of diamond-like and / or amorphous carbon; (xii) optionally rinsing the support after step (xi), for example with an organic solvent, for example acetone and / or absolute ethanol; (xiii) optionally drying the support after step (xi) or (xii) with an inert gas, such as Ar, N2, or any mixture thereof; and (xiv) Binding a biomolecule to the activated surface of at least one sample spot, wherein the support is ready to be used for de novo determination of a single molecular event. DETAILED DESCRIPTION OF THE INVENTION

[0081] Detailed Description The present disclosure relates to a support comprising a substrate coated with a layer of diamond-like carbon and / or amorphous carbon (hereinafter also referred to as "carbon film"). In some embodiments, the support is a substantially planar support, i.e., does not contain any plateaus or depressions of about 1000 nm or more or about 100 nm or more. In further embodiments, the support is a structured support, for example, a substrate having a surface area of ​​about 5 x 10 -24 Liter ~ 1 x 10 -15 The support may contain wells or other depressions with a volume of up to 1 liter, or pillars with a height of approximately 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 generation of a single molecular event on a sample spot present on the support.

[0082] In some 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 region and / or radiation in the near-infrared region. In some embodiments, the substrate comprises a material having an absolute refractive index of at least 1.01 in the visible region, e.g., from about 1.5 to about 3, or from about 1.5 to about 4 in the near-infrared region. In further embodiments, the substrate is an optically opaque material, e.g., a metal or a metalloid such as silicon.

[0083] In certain embodiments, the substrate comprises a non-conductive material. Specific examples include glass, quartz, plastic, metal oxide-based materials, such as glass, silica, or silicon dioxide-based materials such as quartz, or composites containing such materials. In further embodiments, the substrate comprises a conductive material, such as an optically transparent material such as indium tin oxide.

[0084] Typically, the substrate has a thickness of about 10 μm to about 5 mm, particularly about 20 μm to about 2 mm.

[0085] In accordance with the present disclosure, the substrate is coated with a layer of carbon film, which in some embodiments forms a continuous layer on the surface except in the area of ​​at least one sample spot.

[0086] Carbon films can be deposited on substrates by physical vapor deposition, chemical vapor deposition (CVD), or atomic layer deposition (ALD) techniques. In CVD, a surface is exposed to volatile gases containing precursors of the desired deposition material. The precursors are chemically decomposed (typically at high temperatures, although numerous variations of CVD exist in which additional factors such as electromagnetic fields play a role), and the desired material is deposited on the surface while the remaining volatile components are removed by a vacuum pump. CVD films with thicknesses ranging from a few nanometers to microns are achievable. In ALD, films are deposited one atomic layer at a time. The deposition process first involves exposing the surface to linker molecules that form bonds to the surface. A "primer material" (e.g., water) is introduced, making the linker available for reaction with the desired deposition material. The desired deposition material is then introduced, typically from the same type of precursor used in CVD. This bonds with the linker molecules, resulting in the formation of a single atomic layer in a self-limiting manner. The film is then built up by the above iteration. Due to the chemical interactions involved in this process, ALD is more limited than CVD in terms of the materials that can be deposited in this manner, and it can be material-selective. It is also well-suited for the formation of films with thicknesses ranging from a few nanometers to tens of nanometers.

[0087] In some embodiments, the carbon film is fluorinated. Fluorine atoms can be introduced by known procedures, such as by CVD during DLC ​​deposition and / or by exposure to a fluorine-containing plasma from a fluorocarbon-based gas, such as C4F8, CHF3, NF3, or SF6.

[0088] In certain embodiments, the carbon film, including the fluorinated carbon film, has a thickness of about 0.3 nm to about 200 μm, particularly about 3 nm to about 9 nm, about 5 nm to about 100 μm, about 10 nm to about 100 μm, and more particularly about 1 μm to about 50 μm.

[0089] Described herein are surfaces that are selectively coated with a carbon film, resulting in a protein-resistant region, and at least one sample spot adapted to attach a biological moiety, e.g., a biomolecule. In some embodiments, the support comprises a plurality of sample spots, e.g., at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000 or more sample spots. In some embodiments, the support comprises 10 6 Up to 10 pieces 9 The sample may include one or more sample spots.

[0090] 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, including alloys or mixtures of multiple different metals. For example, metals that can be attached to sulfur-containing moieties, such as thiols or disulfides, or metals that can be attached to chelating moieties, 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 combination thereof that includes at least two of the metals.

[0091] 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 moieties, such as phosphonic acid or phosphonate esters, or metal oxides that can be attached to chelating moieties, such as polyhistidine tags, are suitable. Specific examples of suitable metal oxides include TiO2 and NiO.

[0092] In a further embodiment, the sample spot comprises or consists of at least one non-conductive material.

[0093] Sample spots can be prepared by metal deposition, where the metal is evaporated onto a substrate covered with a grid mask, which can be fabricated 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 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, by precise pipetting of the particles onto the substrate, particularly on substrates with flat surfaces.

[0094] The sample spots can have a size suitable for attachment of biological moieties such as biomolecules, cell fractions, organelles, or cells, e.g., prokaryotic or eukaryotic cells, etc. In certain embodiments, the sample spots have a diameter of about 1 nm to about 100 μm, particularly about 2 nm to about 50 μm.

[0095] In certain embodiments, the sample spots have a size suitable for attachment of a single biomolecule, in these embodiments, the sample spots have a diameter of about 1 nm to about 100 nm, particularly about 2 nm to about 50 nm, and more particularly about 5 nm to about 20 nm.

[0096] In some embodiments, the sample spot is a separate object 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, wherein 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 nanometers, particularly about 100 pm to about 20 nm, and more particularly about 500 pm to about 10 nm, for example, about 2 nm.

[0097] There are several options for the placement of the sample spot, carbon layer and substrate.

[0098] In some embodiments, the top surface of the sample spot is flush with the surrounding carbon film. In some embodiments, the top surface of the sample spot is above the surrounding carbon film. In some embodiments, the top surface of the sample spot is below the surrounding carbon film. The distance between the top surface of the sample spot and the carbon film can be in the range of 0.1 nm to 500 nm.

[0099] In some embodiments, the sample spot extends through the carbon layer, so that its lower surface is in direct contact with the surface of the substrate.In some embodiments, the sample spot does not extend through the carbon layer, so that its lower surface is in direct contact with the surface of the carbon layer.In some embodiments, the sample spot is placed on a pillar etched into a planar substrate, where a layer of diamond-like carbon and / or amorphous carbon coats the sidewalls of the pillar and the surface of the planar substrate.

[0100] The sample spot is adapted to attach biological parts, such as biomolecules, cell fractions, organelles, or cells, such as prokaryotic cells or eukaryotic cells.In some embodiments, the sample spot is adapted to attach a single biomolecule, for example, by covalent or non-covalent attachment.The biomolecule can be selected from polypeptides, nucleic acids, carbohydrates, and any combination thereof.In some embodiments, the biomolecule is a nucleic acid polymerizing enzyme, such as RNA polymerase or DNA polymerase, or a nucleic acid degrading enzyme, such as exonuclease.

[0101] In certain embodiments, biological moieties such as biomolecules are directly attached to the sample spots, for example, by providing the biological moieties with anchor groups suitable for covalent or non-covalent attachment to the surface of the sample spots, particularly sulfur- or phosphorus-containing groups such as thiol groups -SH, substituted thiol groups -SR, where R is an organic residue such as a C1-C4 alkyl group or a disulfide group -SS-. Yet another alternative is immobilization in which the binding of biological moieties such as biomolecules can be mediated by reactive silane groups on the silica surface.

[0102] In some embodiments, biological moieties, such as biomolecules, are indirectly attached to the sample spots, e.g., by non-covalent high-affinity linkage to a coating on the surface of the sample spots. In these embodiments, biological moieties, such as biomolecules, can be provided with reactive tags, e.g., biotin, haptens, poly(histidine) tags, or carbohydrate groups, that can form high-affinity linkages with complementary reactive moieties, e.g., streptavidin, antibodies, lectins, etc., attached to the surface of the sample spots.

[0103] In some embodiments, immobilizing biological moieties on a support can include the coupling reaction between two bioorthogonal reactive groups, i.e., groups that do not exist in the biomolecule to be attached to the sample spot.In some embodiments, the coupling reaction is a click reaction, for example, the reaction between an azide group and an alkyne group, for example, a terminal alkyne group or a strained alkyne group, for example, a cyclooctyne group.In some embodiments, the reaction product of the coupling reaction comprises a triazole group.

[0104] In certain embodiments, a biological moiety, such as a biomolecule having a bioorthogonal group, e.g., an azide group or an alkyne group, can be provided that can form a covalent linkage with a complementary bioorthogonal group attached to the surface of the sample spot.

[0105] The supports disclosed herein can be manufactured by different procedures, which are outlined below.

[0106] In one embodiment, the preparation of the support comprises: (i) providing a substrate containing at least one sample spot on its surface; and (ii) Coating the surface of the substrate with a layer of carbon film, for example by physical or chemical vapor deposition (CVD) or by atomic layer deposition (AVD). wherein the material of the sample spots is selected to be insensitive to coating in step (iii), whereby the layer of diamond-like and / or amorphous carbon does not extend over the at least one sample spot.

[0107] According to this embodiment, examples of suitable materials for the sample spots include, but are not limited to, Au, Cu, and Ni, as described in [7] and

[11] .

[0108] In one embodiment, the preparation of the support comprises: (i) coating the surface of the substrate with a carbon film, for example by physical or chemical vapor deposition (CVD) or by atomic layer deposition (AVD); and (ii) depositing at least one sample spot on the surface of said layer; Includes:

[0109] The supports of the present disclosure are suitable for analyzing events occurring on sample spots, where the events are associated with the emission of electromagnetic radiation from the sample spots. In certain embodiments, the events include reactions of biological moieties associated with the emission of characteristic electromagnetic radiation. In certain embodiments, the events are single molecular events.

[0110] In certain embodiments, the support of the present disclosure is suitable for analyzing events, such as single molecular events occurring on at least one sample spot, particularly suitable for separately analyzing multiple single molecular events each occurring on at least one sample spot, and even more particularly suitable for separately analyzing multiple single molecular events in parallel.In certain embodiments, the single molecular event comprises the determination of a nucleic acid sequence.

[0111] A further embodiment relates to a method for analyzing an event, including: (iii) providing the support; (iv) immobilizing a biological moiety, in particular a biomolecule, on at least one sample spot of the support; and (v) Analyzing events associated with said biological moieties, in particular events associated with said biomolecules, by detecting electromagnetic radiation from said sample spots.

[0112] In certain embodiments, the event is a single molecular event and the biomolecule is a single biomolecule.

[0113] In certain embodiments, the single molecule event comprises the sequence analysis of a single nucleic acid molecule.

[0114] A further embodiment relates to a device for analyzing an event, comprising: (iv) a support as described above adapted to immobilize a biological moiety, in particular a biomolecule, on at least one sample spot of the support; (v) a means for directing radiation to at least one sample spot on the support; and (vi) means for analyzing events on said at least one sample spot by detecting electromagnetic radiation from said spot;

[0115] In certain embodiments, the event is a single molecular event and the biomolecule is a single biomolecule.

[0116] In certain embodiments, the device is adapted for sequence analysis of a single nucleic acid molecule.

[0117] Methods and devices for analyzing single molecular 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.

[0118] For the analysis of single molecular events, biomolecules are placed in sample spots on the support, where they come into contact with a sample solution containing free reaction partners, thereby defining one or more reaction spaces, particularly at least 100, at least 1000, or at least 10,000, and up to 10 6 The molecules can be analyzed on a single support, for example a single planar support.

[0119] The nucleic acid molecules whose sequences are to be determined 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 involve the use of 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 pieces or 10 7 This allows for the parallel sequencing of more than two different nucleic acid molecules.

[0120] The nucleic acid molecule to be sequenced can be linear or circular, for example, a single-stranded nucleic acid molecule 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 carried out according to known protocols, for example, by ligation using DNA or RNA ligase.In some embodiments, adapter and / or identifier molecule, i.e., nucleic acid molecule of known sequence, can be linked to nucleic acid molecule.

[0121] Sequencing can involve nucleic acid extension and / or nucleic acid degradation. The sequencing process involves one or more sequencing cycles.

[0122] The nucleic acid synthesizing enzyme molecule can extend a primer annealed to a nucleic acid template molecule. Primer extension can be achieved by gradually 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.

[0123] 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 forms, T7 DNA polymerase, T5 DNA polymerase, RB69 polymerase, and others.

[0124] Nuclease molecules can gradually cleave individual nucleotide building blocks from nucleic acid molecules. Preferably, exonucleases, more preferably single-stranded exonucleases, are used that degrade in the 3' to 5' or 5' to 3' direction. Particularly preferred exonucleases are 3' to 5' exonucleases such as E. coli exonuclease I and E. coli exonuclease III, and 5' to 3' exonucleases such as T7 exonuclease, E. coli exonuclease II, and E. coli exonuclease VIII. Furthermore, the exonuclease activity of various polymerases, such as the Klenow fragment, Taq polymerase, or T4 polymerase, can be used.

[0125] 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 annealed to or capable of annealing 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, which 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 allow 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 nucleic acid synthesizing enzyme molecule, for example, by incorporating a nucleotide analog building block and / or a linkage between the nucleotide building blocks that is stable against degradation. In other embodiments, the primer is sensitive to digestion by the nucleic acid synthesizing enzyme molecule.

[0126] The sequence of the primer is selected so that it can effectively anneal to the template molecule under 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 anneal to a known sequence portion of the nucleic acid template molecule.In some embodiments, 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.

[0127] Furthermore, the presence of a nucleotide building block bearing at least one fluorescent labeling group is required. Preferably, each different nucleotide building block (A, G, C, T / U) contains a different fluorescent labeling group.

[0128] The fluorescent labeling group may be selected from known fluorescent labeling groups used to label biopolymers, particularly nucleic acids, such as fluorescein dyes, rhodamines, oxazines, e.g., Evoblue or Gnothis Blue, phycoerythrin, Cy3, Cy5, IR dyes or derivatives thereof.

[0129] A nucleotide building block may have (i) a fluorescent labeling group that remains with 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 with the building block is preferably attached to the α-phosphate group, sugar, and / or nucleobase group.

[0130] In certain embodiments, the fluorescent labeling group remaining with the building block can have a chain length of, for example, up to 15 carbon atoms, preferably 10-12 carbon atoms, and is optionally linked to the nucleic acid base via a linker containing a heteroatom, such as an N, O, or S atom. The fluorescent labeling group that is cleaved when the building block is incorporated into a nucleic acid molecule can be linked to the terminal phosphate group of a polyphosphate building block, including, but not limited to, a hexa-, penta-, tetra-, or triphosphate building block, such as the γ-phosphate group of a triphosphate building block. In some 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, for example, fluorescent groups that can interact with each other by quenching and / or energy transfer can be selected.

[0131] The nucleic acid molecule to be sequenced may contain 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.

[0132] The sequencing procedure may include a step of generating a nucleic acid molecule with incorporated nucleotide building blocks in a primer extension catalyzed by a nucleic acid-synthesizing enzyme 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 may be performed during primer extension and / or degradation.

[0133] Sequencing during primer extension involves the use of nucleotide building blocks with fluorescent labeling groups that are cleaved from the building blocks when incorporated into nucleic acid molecules. In this case, the time-dependent fluorescence change caused by the cleavage of the fluorescent labeling group from the nucleotide building blocks can be determined. Sequencing during nucleic acid degradation involves the use of nucleotide building blocks with fluorescent labeling groups that remain with the building blocks when incorporated into nucleic acid molecules. The gradual cleavage of individual nucleotide building blocks from nucleic acid molecules results in a time-dependent change in fluorescence when the labeled nucleotide building blocks are released from the nucleic acid molecule. In some embodiments, sequencing can also be performed during extension and degradation, i.e., when using nucleotide building blocks that have both a fluorescent labeling group that remains with the building block when the building block is incorporated into a nucleic acid molecule and a fluorescent labeling group that is cleaved from the building block. In this embodiment, both fluorescent groups can be the same or different.

[0134] 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 a nucleic acid template molecule, which is catalyzed by a nucleic acid synthesis enzyme molecule, whereby a nucleic acid molecule complementary to the sequence of the nucleic acid template is produced.In the next step, the produced nucleic acid molecule is degraded by a nucleic acid degradation enzyme molecule.

[0135] 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, the 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 has a fluorescent labeling group on a portion of the molecule that is cleaved when the building block is incorporated into the primer, for example, on the γ-phosphate group.

[0136] When nucleotide building blocks are cleaved from the synthesized nucleic acid molecule, a time-dependent change in fluorescence can be determined due to interactions of the fluorescent labeling group incorporated into the nucleic acid chain with neighboring groups, e.g., chemical groups of the nucleic acid, in particular nucleic acid bases such as G, or / and neighboring fluorescent labeling groups, and these interactions result in changes in fluorescence, in particular fluorescence intensity, due to quenching or / and energy transfer processes, compared to the fluorescent labeling groups in their "isolated" form. Cleavage removal of individual nucleotide building blocks changes the overall fluorescence, e.g., fluorescence intensity, of the immobilized nucleic acid chain, and this change is a function of cleavage removal of individual nucleotide building blocks, i.e., a function of time.

[0137] In some 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 paired labeled nucleotide that is free to move during the polymerization process.

[0138] 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. It is preferable to use fluorescent labeling groups that are at least partially quenched when incorporated into a nucleic acid strand, resulting in an increase in fluorescence intensity after the nucleotide building block containing the labeling group or the building block that causes the quenching is removed by cleavage.

[0139] 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 or / and 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 studied nucleic acid strand and can therefore be correlated with the sequence.

[0140] The complete sequence of a nucleic acid molecule can be determined using a mixture of nucleotide building blocks labeled with all four different bases, e.g., A, G, C, and T, or combinations of two or three different bases. Where appropriate, it is also possible to attach a "sequence identifier," i.e., a labeled nucleic acid of known sequence, to the nucleic acid strand under study by an enzymatic reaction, e.g., using a ligase and / or terminal transferase, so that at the start of sequencing, an initially known fluorescence pattern is obtained, and only thereafter a fluorescence pattern corresponding to the unknown sequence under study is obtained.

[0141] Detection preferably involves irradiating the support with light using a laser or other suitable light source to excite the fluorescent labeling groups. One or more laser beams, e.g., an expanded laser beam having a cross-sectional area of ​​about 1 to 20 mm, and / or multiple laser beams can be used. Detection preferably involves multipoint fluorescence excitation by a laser, e.g., by a dot matrix of laser dots generated by diffraction optics (see WO 2002 / 097406) or a quantum well laser.

[0142] The fluorescence emissions of multiple nucleic acid strands can be detected in parallel using a detector matrix, including, for example, an electronic detector matrix such as a CCD camera, a CMOS detector matrix such as a CMOS camera, or an avalanche photodiode matrix. Detection can be performed so 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 substantially orthogonally from the support surface through the reaction space or support body.

[0143] Detection can be performed, for example, by single molecule detection, for example, by fluorescence correlation spectroscopy, which can be performed, for example, by 10 -21 ~10 -10The method involves exposing a very small, preferably confocal, volume element of 1 to excitation light from a laser or other suitable light source, which excites receptors present in the measurement volume, causing them to emit fluorescent light. The fluorescent light emitted from the measurement volume is measured by a photodetector, and the time course of the measured emission is correlated with the concentration of the analyte, thereby making it possible to identify individual molecules within the measurement volume at suitably high dilutions. Details of the procedure and 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.

[0144] Alternatively, or in addition, detection can also be performed by 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 contents of which are incorporated herein by reference. Here, fluorescent molecules are excited within the measurement volume and then detected on a photodetector at a time interval of, for example, ≥ 100 ps. In this way, the background signal generated by the Raman effect can be kept sufficiently low, allowing single molecules to be detected in a substantially interference-free manner.

[0145] A second aspect of the present disclosure relates to a method for cleaning a previously used support, wherein the support is adapted for use in devices and methods for analyzing events, e.g., single molecular events. Unless otherwise specified, features of the first aspect also apply to the second aspect.

[0146] The previously used support as understood herein includes a support on which a previous analysis, for example, a single molecule analysis, has been performed. Thus, the support has attached to it at least one sample spot a biological moiety such as a biomolecule, and is optionally contaminated with additional components, for example, components of the sample on which the analysis was performed. By the method disclosed herein, the biological moiety, for example, the biomolecule attached to the sample spot and any additional components, can be removed from the support without causing substantial damage.

[0147] This aspect includes the reusability of the support. Once the support is used for its desired application, it contains a sample spot on a substrate, e.g., glass, surrounded by a diamond-like carbon and / or amorphous carbon (DLC or α-carbon) film, along with biomolecules or biomolecular debris bound to the sample spot, as well as possible residues from reagents. In some cases, biomolecular debris and reagent residues are also present in the coated areas. To return the surface to the "pristine" condition required for its reuse, the surface must be thoroughly cleaned to remove debris and residue.

[0148] Alkaline solutions are effective in removing nucleic acid deposits, such as DNA or RNA deposits. They are commercially available (e.g., DNA Away) and can be used to treat used supports. Treatment can be performed at approximately 20°C to 25°C or, for example, at elevated temperatures to enhance effectiveness. Acidic / oxidizing solutions, such as "piranha solution," consisting of a mixture of sulfuric acid and hydrogen peroxide, are very effective in removing proteins and protein deposits. These treatments can be performed sequentially, with intermediate rinsing steps as needed. These cleaning steps should leave the surface—the metal sample spot surrounded by a diamond-like and / or amorphous carbon film—substantially untouched.

[0149] An additional cleaning step consisting of exposure to O2 plasma can provide even further cleaning, but may remove some of the DLC or α-carbon film and therefore should be done with care. In some embodiments, the diamond-like carbon and / or amorphous carbon film can then be refluorinated, for example, by exposure to a fluorine-containing plasma, as in the initial film formation.

[0150] In this embodiment, the entire DLC or α-carbon film is removed by etching in O2 plasma, essentially exposing the underlying glass substrate and metal sample spots. The substrate can then be baked at high temperature to ensure that the metal sample spots provide a fresh surface for biomolecule attachment. If the sample spots are nanoparticles, they can be melted and reformed in this process. Formation of a new DLC or α-carbon film is then achieved as described above.

[0151] An embodiment of the second aspect relates to a method for cleaning a previously used substrate, comprising subjecting the used substrate to (a) treatment with an alkaline solution, (b) treatment with an acidic / oxidizing solution, optionally (c) exposure to an O2 plasma, and (d) optionally exposure to a fluorine-containing plasma.

[0152] Specific embodiments of the second aspect include: (i) Providing a support comprising a substrate and at least one sample spot on a surface of the support, wherein a biological moiety, e.g., a biomolecule, is attached to the at least one sample spot. (ii) subjecting the support from step (i) to treatment with an alkaline solution containing, for example, an alkaline phosphate, such as potassium phosphate, and optionally a surfactant and / or a chelating agent, wherein the treatment is carried out at an elevated temperature, in particular at the boil; (iii) optionally rinsing the support after step (ii), e.g., with water; (iv) subjecting the support after step (ii) or (iii) to treatment with an acidic / oxidizing solution comprising a strong inorganic acid, e.g., sulfuric acid, and a peroxide, e.g., hydrogen peroxide; (v) optionally rinsing the support after step (iv), e.g., with water and / or an anhydrous organic solvent, e.g., ethanol; (vi) optionally drying the support after step (iv) or (v) with an inert gas, such as Ar, N2, or any mixture thereof; (vii) subjecting the support after step (iv), (v) or (vi) to a plasma treatment, in particular an O2 plasma treatment. The present invention relates to a method for cleaning a previously used substrate, comprising:

[0153] In certain embodiments of this aspect, the support to be cleaned comprises a substrate and at least one sample spot on a surface of the substrate, wherein the substrate is at least partially coated with a layer of diamond-like and / or amorphous carbon, and wherein the layer of diamond-like and / or amorphous carbon does not extend over the at least one binding spot.

[0154] In a further embodiment of this aspect, the support to be cleaned comprises a substrate and at least one sample spot on a surface of the substrate, wherein the substrate is at least partially coated with a layer of an organic passivating material, e.g., a poly(ethyleneoxy) group-containing passivating material, and wherein the layer of organic passivating material does not extend over the at least one sample spot.

[0155] In some embodiments of this aspect, biological moieties, such as biomolecules, are attached to the cleaned support after step (vii). Furthermore, the support surface surrounding the sample spot can be regenerated to inhibit adhesion of biomolecules and / or other sample components, for example, by treating it with a passivation reagent that inhibits adhesion of biomolecules, such as proteins. In some embodiments, passivating the substrate can include treatment with a PEGylation reagent, such as an alkoxy-PEG silane, for example, a methoxy-PEG silane. Alternatively, regenerating can include coating the surface of the support with a fresh layer of diamond-like and / or amorphous carbon. The support is then ready for use in a new analysis of an event, for example, a single molecular event. The regeneration step is typically performed before the step of attaching biological moieties, such as biomolecules. When using the carbon film-coated support described above in the first aspect, the regeneration step is not necessary in some embodiments.

[0156] In some embodiments, the attachment of a biological moiety, e.g., a biomolecule, is by: (viii) activating the surface of at least one sample spot, e.g., subjecting the support after step (vii) to treatment with a thiol reagent; (ix) optionally rinsing the support after step (viii) with an organic solvent, such as absolute ethanol; (x) optionally drying the support after step (viii) or (ix) with an inert gas, such as Ar, N2, or any mixture thereof; (xi) optionally regenerating the surface of the support, e.g., passivating the substrate by treatment with a PEGylation reagent such as an alkoxy PEG silane, e.g., a methoxy PEG silane, or by coating the surface of the support with a layer of diamond-like and / or amorphous carbon; (xii) optionally rinsing the support after step (xi), for example with an organic solvent, for example acetone and / or absolute ethanol; (xiii) optionally drying the support after step (xi) or (xii) with an inert gas, such as Ar, N2, or any mixture thereof; and (xiv) binding a biomolecule to the activated surface of at least one sample spot, where the support is ready for use in a new analysis of a single molecular event. Includes:

[0157] Furthermore, the present disclosure will be described in detail by reference to the following specific embodiments. [Brief explanation of the drawings]

[0158] [Figure 1] 1 (Prior Art) shows an optically transparent substrate (e.g., quartz; fused silica; or glass) coated with a layer of diamond-like carbon (DLC) deposited by chemical vapor deposition (CVD) with a thickness ranging from about 1 nm to 10 μm. After deposition, the DLC film is exposed to a fluorine-containing plasma. [Figure 2] 2 (Prior Art) shows an optically transparent substrate (e.g., quartz; fused silica; or glass) coated with a layer of amorphous carbon (DLC) having a thickness ranging from about 1 nm to 100 nm, deposited by atomic layer deposition (ALD). After deposition, the film is exposed to a fluorine-containing plasma. [Figure 3]Embodiments 3 and 4 (present disclosure) of Figures 3A and 3B show optically transparent substrates (e.g., quartz; fused silica; or glass) coated with DLC deposited by CVD or ALD, with thicknesses ranging from 1 nm to 10 μm. After deposition, the DLC is coated with photoresist, optionally after treatment with oxygen plasma. The photoresist is then patterned by lithographic techniques (e.g., optical or electron beam lithography, or any other suitable technique) and developed to form a stencil in the resist with the desired pattern. According to Figure 3A, an underlying metal or metal oxide layer is present. Openings are formed in the carbon film by etching, exposing selected areas of the underlying film. These openings can form sample spots. The carbon film is exposed to a fluorine-containing plasma. According to Figure 3B, metal or metal oxide features are fabricated on the top surface of the carbon film, for example, by conventional lift-off patterning. The carbon film is exposed to a fluorine-containing plasma. [Figure 4] Embodiment 5 (present disclosure) in Figures 4A and 4B shows an optically transparent substrate (e.g., quartz; fused silica; or glass) on which metal spots have been patterned using a lithographic patterning process, resulting in a pattern of metal spots positioned at predetermined locations on the substrate's surface. The metal is a non-adhesive metal, i.e., it does not react with DLC (or, in the case of ALD, with the ALD linker). Au, Cu, and Ni are examples of such metals. [7] The surface is then selectively coated with DLC. Because DLC does not bond to the metal, carbon does not deposit on the metal, but only on the glass. The substrate is exposed to a fluorine-containing plasma.

[0159] Embodiment 6 (the present disclosure) includes a solid substrate (e.g., glass) and at least one sample spot or multiple sample spots on the surface of the substrate, where the sample spots are designed to accommodate a single biomolecule of interest or at most a small number of biomolecules of interest, e.g., up to five biomolecules of interest.

[15] In certain embodiments, the sample spots have lateral and vertical dimensions of less than 10 nm but not more than 20 nm, e.g., in a size range of about 2 nm to about 20 nm, or about 5 nm to about 20 nm. The sample spots can be formed by directly forming thin metal platelets of the desired metal material using lithographic techniques.

[16] Or they can take the form of nanoparticles produced using a combination of lithographic patterning and post-processing to produce spherical or spheroidal metal particles of the desired size and shape. [15, 17-19]

[0160] In this embodiment, the sample spots are constructed on the substrate in a predetermined spatial arrangement, such that the position of every single biomolecule at the sample spot is known. This facilitates subsequent detection of biomolecular interactions of interest by a detector system (e.g., fluorescence). The spatial arrangement can be an ordered pattern, such as a Cartesian lattice, a hexagonal array, or a Fibonacci map / layout, with interspot distances ranging from 20 nm to 10 μm or more, such that the position of each sample spot on the substrate is known within an accuracy of about + / - 5 nm, + / - 10 nm, or + / - 25 nm. Such an arrangement can be achieved by nanolithographic patterning techniques (e.g., electron beam nanolithography, nanoimprint nanolithography, extreme ultraviolet nanolithography, etc.) and, if necessary, additional processing.

[0161] The sample spots in this embodiment are composed of metal and can be fabricated by, for example, the process described in [15, 17-19] or any other lithographic [15-19] or lithography-guided self-assembly process capable of producing metal features in the size range of less than 20 nm. Attachment of the biomolecule of interest is achieved by one of several techniques described in the literature (see, for example, [15, 17-19]).

[0162] To ensure that the biomolecule of interest is confined to the sample spot and not bound or adsorbed to the surrounding area, the surrounding substrate area is coated with a layer of diamond-like carbon and / or amorphous carbon (DLC or α-carbon) film with a thickness less than the height of the sample spot, thereby leaving the sample spot exposed for further reaction with the selected biomolecule. In some embodiments, the layer thickness is at least about 1 nm and at most about 0.1 nm less than the height of the sample spot, for example, a layer thickness of 1 to 4.9 nm for a sample spot height of 5 nm, or a layer thickness of 1 to 19.9 nm for a sample spot height of 20 nm.

[0163] The DLC or α-carbon film can be fluorinated either in situ during deposition or after deposition (eg, by exposure to a fluorine-containing plasma).

[0164] This selective deposition can be achieved by either one of two means: (i) By choosing deposition conditions and / or deposition precursors, DLC or α-carbon films grow selectively on glass and not on the sample spot material (e.g., gold). This approach is particularly relevant to atomic layer deposition (ALD), where precursors can be chosen to selectively bond to the glass substrate and not to the sample spot [20, 21], ensuring that DLC or α-carbon films [22, 23] grow only in the area surrounding the sample spot, as in Figure 5a, b. (ii) Alternatively, or additionally, the sample spots can be protected by, for example, a self-assembled monolayer (SAM) that adheres only to the spots

[21] . The SAM prevents the growth of DLC or α-carbon films on the spots, as in Figure 5c-e.

[0165] EMBODIMENT 7 (This disclosure) follows the basic layout of embodiment 6, except for the method of forming the DLC or α-carbon film surrounding the sample spot. In this embodiment, the DLC or α-carbon film is deposited on the entire surface to a thickness large enough to cover the sample spot, as in Figures 6a and 6b. The deposited film is then etched back, for example, by reactive ion etching in oxygen plasma, to expose the top surface of the sample spot, while a reduced-thickness DLC or α-carbon film remains surrounding the sample spot, as in Figure 6c.

[0166] For large-area spots and relatively thin deposited films, this process may result in complete removal of the film outside the sample spot due to small or even zero thickness differences above and outside the sample spot. For nanometer-sized spots and relatively thin deposited films, e.g., films with thicknesses of about 100 nm or less, especially about 20 nm or less, the etching process may be well-tuned to expose the top surface of the sample spot without removing the deposited film outside these regions (Figure 6c). DLC or α-carbon films may be fluorinated either in situ during deposition or after deposition (e.g., by exposure to a fluorine-containing plasma).

[0167] References 1. Bernhard, C., et al., Repelling and ordering: the influence of poly(ethylene glycol) on protein adsorption.Physical Chemistry Chemical Physics, 2017. 19(41): p. 28182-28188. 2. Pan, H., et al., A simple procedure to improve the surface passivation for single molecule fluorescence studies. Physical Biology, 2015. 12(4): p. 045006. 3. Park, S.R., et al., A Single-Molecule Surface-Based Platform to Detect the Assembly and Function of the Human RNA Polymerase II Transcription Machinery. Structure, 2020. 28(12): p. 1337-1343.e4. 4. Wong, I. and C.-M. Ho, Surface molecular property modifications for poly(dimethylsiloxane) (PDMS) based microfluidic devices. Microfluidics and Nanofluidics, 2009. 7(3): p. 291. 5. Yonezawa, K., et al., Evaluation of Antibacterial and Cytotoxic Properties of a Fluorinated Diamond-Like Carbon Coating for the Development of Antibacterial Medical Implants. Antibiotics (Basel), 2020. 9(8). 6. Horikawa, A.M., Shunto; Hasebe, Terumitsu; Matsumoto, Tomohiro; Tanaka, Minoru; akahashi, Koki; Suzuki, Tetsuya, Fluorine-incorporated amorphous carbon coating inhibits adhesion of blood cells to biomaterials. Sensors and Materials, 2017. 29(6): p. 9. 7. Outka, D.A.H., Wen L.; Boehme, D. R.; Yang, N. Y. C.; Ottesen, D. K.; Johnsen, H. A., Compilation of diamond-like carbon properties for barriers and hard coatings, in Sandia Report. 1994. 8. Rajak, D.K., et al., Diamond-Like Carbon (DLC) Coatings: Classification, Properties, and Applications.Applied Sciences, 2021. 11(10): p. 4445. 9. Robertson, J., Diamond-like amorphous carbon. Materials Science and Engineering: R: Reports, 2002. 37(4): p. 129-281. 10. Bendavid, A., et al., The properties of fluorine-containing diamond-like carbon films prepared by pulsed DC plasma-activated chemical vapour deposition. Diamond and Related Materials, 2010. 19(12): p. 1466-1471. 11. Schvartzman, M., et al., Plasma fluorination of carbon-based materials for imprint and molding lithographic applications. Applied Physics Letters, 2008. 93(15): p. 3. 12. Schvartzman, M., et al., Fluorinated diamondlike carbon templates for high resolution nanoimprint lithography.Journal of Vacuum Science & Technology B, 2008. 26(6): p. 2394-2398. 13. Carvalho, I., et al., Overview on the Antimicrobial Activity and Biocompatibility of Sputtered Carbon-Based Coatings. Processes, 2021. 9(8): p. 1428. 14. Hasebe, T., et al., Fluorine doping into diamond-like carbon coatings inhibits protein adsorption and platelet activation. Journal of Biomedical Materials Research Part A, 2007. 83A(4): p. 1192-1199. 15. Cai, H., et al., Molecular Occupancy of Nanodot Arrays. ACS Nano, 2016. 10(4): p. 4173-4183. 16. Cherniavskaya, O., et al., Fabrication and surface chemistry of nanoscale bioarrays designed for the study of cytoskeletal protein binding interactions and their effect on cell motility. Journal of Vacuum Science & Technology B, 2005. 23(6): p. 2972-2978. 17. Cai, H., et al., Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering. Nature Nanotechnology, 2018. 13(7): p. 610-617. 18. Penzo, E., et al., Directed Assembly of End-Functionalized Single Wall Carbon Nanotube Segments. Nano Letters, 2015. 15(10): p. 6547-6552. 19. Schvartzman, M., et al., Nanolithographic Control of the Spatial Organization of Cellular Adhesion Receptors at the Single-Molecule Level. Nano Letters, 2011. 11(3): p. 1306-1312. 20. Cao, K., J. Cai, and R. Chen, Inherently Selective Atomic Layer Deposition and Applications. Chemistry of Materials, 2020. 32(6): p. 2195-2207. 21. Oh, I.-K., et al., Role of Precursor Choice on Area-Selective Atomic Layer Deposition. Chemistry of Materials, 2021. 33(11): p. 3926-3935. 22. Choi, T., et al., Hydrogen plasma-enhanced atomic layer deposition of hydrogenated amorphous carbon thin films. Surface and Coatings Technology, 2018. 344: p. 12-20. 23. Xiao, Z., K. Kisslinger, and R. Monikandan, Atomic Layer Deposition of Nanolayered Carbon Films. C, 2021. 7(4): p. 67.

Claims

1. a support comprising a substrate and at least one sample spot on a surface of the support; A support, wherein the substrate is at least partially coated with a layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon, and wherein the layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon does not extend over at least one bonding spot.

2. The support of claim 1 which is at least substantially planar.

3. 10. The support of any one of the preceding claims, comprising a plurality of sample spots.

4. 10. The support of any one of the preceding claims, wherein at least one sample spot comprises at least one metal or metal oxide.

5. 10. The support of any one of the preceding claims, wherein at least one sample spot has a diameter of about 1 nm to about 100 μm, in particular a diameter of about 2 nm to about 50 nm, and more particularly a diameter of about 5 nm to about 20 nm.

6. (i) at least one sample spot has a top surface distal to the substrate and that top surface is level with the surrounding layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon; (ii) at least one sample spot has a top surface distal to the substrate, and the top surface of the sample spot is above a surrounding layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon; or (iii) at least one sample spot has a top surface distal to the substrate, and the top surface of the sample spot is below a surrounding layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon; A support according to any one of the preceding claims.

7. (i) at least one sample spot extends through the carbon layer so that its lower surface is in direct contact with the surface of the substrate; or (ii) at least one sample spot does not extend through the carbon layer, such that its lower surface is in direct contact with the surface of the carbon layer; or (iii) at least one sample spot is located on a post etched into a planar substrate, wherein a layer of diamond-like and / or amorphous carbon coats the sidewalls of the post and the surface of the planar substrate; A support according to any one of the preceding claims.

8. 10. The support of claim 1, wherein the layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon has a thickness of from about 0.3 nm to about 200 μm, in particular from about 3 nm to about 9 nm, from about 5 nm to about 100 μm, from about 10 nm to about 100 μm, or from about 1 μm to about 50 μm.

9. 10. A support according to any one of the preceding claims, wherein a biological moiety, in particular a single biomolecule, is attached to at least one of the sample spots, wherein the biological moiety is in particular a nucleic acid-polymerizing enzyme, such as an RNA polymerase or a DNA polymerase.

10. The support used was treated with (a) alkaline solution, (b) acidic / oxidizing solution, and (c) O 2 d) optionally exposing the support to a fluorine-containing plasma; and d) optionally exposing the support to a fluorine-containing plasma.

11. Use of a support according to any one of claims 1 to 10 for analyzing an event, wherein the event is associated with the emission of electromagnetic radiation from a sample spot, in particular a single molecule event comprising the determination of a nucleic acid sequence occurring on at least one sample spot, in particular for separately analyzing a plurality of single molecule events each occurring on at least one sample spot, and more particularly for separately analyzing a plurality of single molecule events in parallel.

12. 1. A method for analyzing an event, comprising: (i) providing a support according to any one of claims 1 to 10; (ii) immobilizing a biological moiety on at least one sample spot of the support; and (iii) analyzing events associated with the biological moiety by detecting electromagnetic radiation from the sample spot; A method comprising:

13. The method of claim 12 , wherein the event comprises sequence analysis of a single nucleic acid molecule.

14. 1. A device for analyzing an event, comprising: (i) a support according to any one of claims 1 to 10; (ii) a means for directing radiation to at least one sample spot on the support; and (iii) means for analyzing events on said at least one sample spot by detecting electromagnetic radiation from said spot; Including, the device.

15. 15. The device of claim 14 adapted for sequence analysis of a single nucleic acid molecule.

Images