Selective region coated anti-protein surfaces

Abstract

The present disclosure relates to a carrier comprising an optically transparent substrate and at least one sample point on a surface of the carrier, a device for determining a single molecular event comprising the carrier, and a method for determining a single molecular event in which the carrier is used.

Description

Technical Field

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

[0002] The development of biomedical devices and systems, from biosensors to drug delivery, relies heavily on the use of protein-resistant surfaces. This is also an absolute requirement for single-molecule studies of biomolecular interactions, where any signal from nonspecifically bound proteins on the test device surface interferes 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 attached to the underlying substrate and arranged in a thin, brush-like layer in a variety of configurations [1]. However, PEG passivation is often flawed, and a number of techniques have been developed to improve its coverage and effectiveness [2-4]. Other known organic passivation films have similar drawbacks [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 strong, with mechanical strength far exceeding that of organic layers. They are relatively immune to environmental or ambient conditions (e.g., they do not have to be stored in water), properties that also make them suitable for repeated use, and have been found to resist protein adhesion, and are considered excellent candidates for coatings for biomedical devices such as implants [5,6]. DLC films have outstanding mechanical and tribological properties [7,8] and have low surface energy. They have found use as anti-wear coatings in a variety of applications [9].

[0005] A further reduction in surface energy, and therefore wettability, can be achieved by introducing fluorine into the film. Fluorine can be introduced during DLC deposition by CVD

[10] or by exposure to a fluorine-containing plasma from a fluorocarbon-based gas, such as C4F8, CHF3, NF3, or SF6. The fluorine atoms are present at the atomic spacing of the surface

[10] or within the atomic spacing of the surface [11, 12]. The result is a highly hydrophobic (or superhydrophobic) surface [11, 12] that resists the adhesion of many substances and, in particular, has been shown to be extremely resistant to the adhesion of cells and biomolecules [6, 13, 14].

[0006] However, the present inventors were not aware of the use of optically transparent surfaces coated with an inorganic carbon layer in single molecule studies involving optically detected biomolecular interactions in a reaction space located on the coated support. Summary of the Invention

[0007] Support coated with carbon film

[0008] In a first aspect, the present disclosure relates to supports regioselectively coated with carbon films and apparatus and methods for analyzing events associated with the emission of electromagnetic radiation, such as single molecule events, wherein the supports are used.

[0009] According to this aspect, the carrier includes a substrate having a surface coated with a film of diamond-like carbon (DLC) and / or amorphous carbon in a regioselective manner, so that its anti-protein and other biological parts adhere in the coated region while allowing the biological part to be selectively connected to the uncoated region. 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 treating with a fluorine-based plasma. The carbon film is mechanically strong and chemically stable, and it can withstand multiple use sequences with minimal cleaning and / or reactivation steps. Therefore, the surface coated with carbon can replace the surface processed with conventional protein repellent reagents such as polyethylene glycol (PEG).

[0010] In a first embodiment of this aspect, the present disclosure relates to a carrier comprising a substrate and at least one sample site on a surface of the carrier, 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 the at least one sample site. In a specific embodiment, the substrate is an optically transparent substrate.

[0011] Another embodiment relates to the use of the above-described carrier for analyzing an event at at least one sample site, wherein the event is associated with the emission of electromagnetic radiation. In a specific embodiment, the event is a single molecule event.

[0012] In specific embodiments, the carbon layer is a layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon.

[0013] In another embodiment, the carrier is reusable after undergoing a cleaning procedure in which biological moieties attached to at least one sample site are removed. When the carbon layer is a layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon, the cleaning efficiency of the used carrier can be improved.

[0014] Another embodiment relates to a method of making the above-described carrier, comprising:

[0015] (i) providing a substrate comprising at least one sample site on a surface of the substrate, and

[0016] (ii) coating the surface of the substrate with a layer of diamond-like carbon and / or amorphous carbon,

[0017] The material of the sample sites is selected such that it is not susceptible to the coating in step (iii), such that the layer of diamond and / or amorphous carbon does not extend over at least one sample site.

[0018] Another embodiment relates to a method of making the above-described carrier, comprising:

[0019] (i) coating the surface of the substrate with a layer of diamond-like carbon and / or amorphous carbon, and

[0020] (ii) depositing at least one sample site on the surface of the layer.

[0021] Another embodiment relates to a method of making the above-described carrier, comprising:

[0022] (i) coating the surface of a substrate with a layer of diamond-like carbon and / or amorphous carbon,

[0023] (ii) removing the layer in selected areas of the surface, and

[0024] (iii) depositing at least one sample spot in the selected area from which the layer has been removed.

[0025] Another embodiment relates to a method for analyzing an event, the method comprising:

[0026] (i) providing the above-mentioned vector,

[0027] (ii) immobilizing a biological moiety, in particular a biomolecule, on at least one sample site of the carrier, and

[0028] (i) analyzing events associated with said biological moiety, in particular events associated with said biomolecule, by detecting electromagnetic radiation from said sample site.

[0029] In specific embodiments, the event is a single molecular event, and the biomolecule is a single biomolecule.

[0030] Another embodiment relates to an apparatus for analyzing an event, the apparatus comprising:

[0031] (i) the above-mentioned carrier, which is suitable for immobilizing biological moieties, in particular biomolecules, on at least one sample site of the carrier,

[0032] (ii) means for irradiating at least one sample site of the carrier with radiation, and

[0033] (iii) means for analyzing events at said at least one sample site by detecting electromagnetic radiation from said site.

[0034] In specific embodiments, the event is a single molecular event, and the biomolecule is a single biomolecule.

[0035] Cleaning of previously used carriers

[0036] A second aspect described herein is a method for cleaning a previously used carrier suitable for use in an apparatus and method for analyzing an event, such as a single molecule event, wherein the event is associated with the emission of electromagnetic radiation. According to this aspect, the carrier preferably comprises a carbon layer, wherein the carbon layer is a layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon.

[0037] In a specific embodiment, the cleaning procedure comprises 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 (d) optionally exposure to a fluorine-containing plasma.

[0038] Unless otherwise stated, the features of the first aspect also apply to the second aspect.

[0039] An embodiment of the second aspect relates to a method of cleaning a previously used carrier, the method comprising:

[0040] (i) providing a carrier comprising a substrate and at least one sample site on a surface of the carrier, wherein the biological moiety is attached to the at least one sample site;

[0041] (ii) subjecting the support from step (i) to a treatment with an alkaline solution, wherein the treatment is carried out at an elevated temperature;

[0042] (iii) optionally washing the support after step (ii);

[0043] (iv) subjecting the support after step (ii) or (iii) to treatment with an acidic / oxidizing solution comprising a strong mineral acid and a peroxide;

[0044] (v) optionally washing the support after step (iv);

[0045] (vi) optionally drying the support after step (iv) or (v) with an inert gas;

[0046] (vii) optionally subjecting the support after step (iv), (v) or (vi) to a plasma treatment, for example with an O2 plasma and / or with a fluorine-containing plasma.

[0047] In specific embodiments, the substrate is an optically clear substrate.

[0048] In another embodiment of this aspect, after step (vii), the biological part, such as a biomolecule, is attached to the cleaned carrier. In addition, in order to inhibit the adhesion of the biological part, such as a biomolecule and / or other sample components, the carrier surface surrounding the sample point can be passivated, i.e., treated with a protein adhesion repellent. Thereafter, the carrier is ready for use in a new analysis of an event, such as a single molecule event.

[0049] Items in the instruction manual

[0050] Hereinafter, the specific items of this specification are described:

[0051] 1. A carrier comprising a substrate and at least one sample site on a surface of the carrier,

[0052] The substrate is at least partially coated with a layer of diamond-like carbon and / or amorphous carbon, and the layer of diamond-like carbon and / or amorphous carbon does not extend over at least one sample site.

[0053] 2. The carrier of item 1, which is at least substantially planar.

[0054] 3. The carrier of item 1 or 2, which is structured.

[0055] 4. The carrier of any of the preceding items, wherein the substrate is optically transparent.

[0056] 5. The carrier of any of the preceding items, wherein the substrate comprises a material having a refractive index of at least 1.01.

[0057] 6. The carrier of any of the preceding items, wherein the substrate comprises a non-conductive material.

[0058] 7. The carrier of any of the preceding items, wherein the substrate comprises a material selected from the group consisting of silicon dioxide, quartz, and glass.

[0059] 8. The carrier of any 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.

[0060] 9. The carrier of any of the preceding items, comprising a plurality of sample sites, such as 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 sites.

[0061] 10. The carrier of any of the preceding items, wherein at least one sample site comprises at least one electrically conductive material, such as a metal including a pure metal or a combination of metals, such as an alloy or a mixture of a plurality of different metals.

[0062] 11. The support according to claim 10, wherein at least one metal is capable of forming a bond with sulfur, for example in the form of a thiol or a disulfide.

[0063] 12. The support of any one of items 10-11, wherein at least the metal has a positive electrochemical potential.

[0064] 13. The support of any one of items 10-12, wherein at least one metal is selected from Au, Cu, Ni, Pt, Pd, Rh, Ir, Os, Ru and any combination comprising at least two of the aforementioned metals.

[0065] 14. The support of any one of items 1 to 9, wherein at least one sample site comprises at least one metal oxide, such as TiO2, NiO or ITO.

[0066] 15. The carrier of any 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.

[0067] 16. The carrier of any 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, more particularly a diameter of about 5 nm to about 20 nm.

[0068] 17. The carrier of any of the preceding items, wherein at least one sample site has an upper face remote from the substrate, and wherein the upper face has a height equal to that of the surrounding layer of diamond-like carbon and / or amorphous carbon.

[0069] 18. The carrier of any one of items 1 to 17, wherein at least one sample site has a top surface remote from the substrate, and wherein the top surface of the sample site is higher than the surrounding layer of diamond-like carbon and / or amorphous carbon.

[0070] 19. The carrier of any one of items 1 to 17, wherein at least one sample site has a top surface remote from the substrate, and wherein the top surface of the sample site is lower than the surrounding layer of diamond-like carbon and / or amorphous carbon.

[0071] 20. A carrier according to any of the preceding items, wherein at least one sample spot has a lower face adjacent to the substrate and a top face remote from the substrate, wherein the distance between the lower face and the top face defines the height of the sample spot, and wherein the height is from about 50 pm to about 500 nm, in particular from about 100 pm to about 20 nm, and more in particular from about 500 pm to about 10 nm, for example about 2 nm.

[0072] 21. The carrier of any of the preceding items, wherein at least one sample site extends through the carbon layer such that its bottom surface is in direct contact with the surface of the substrate.

[0073] 22. The carrier of any one of items 1 to 20, wherein at least one sample site does not extend through the carbon layer such that its bottom surface is in direct contact with the surface of the carbon layer.

[0074] 23. The carrier of any of items 1-20, wherein at least one sample site is located on a post that has been etched into a planar substrate, wherein a layer of diamond-like carbon and / or amorphous carbon coats the sidewalls of the post and the planar surface of the substrate.

[0075] 24. The carrier of any of the preceding items, wherein the layer of diamond-like carbon and / or amorphous carbon has a thickness of about 0.3 nm to about 200 μm, in particular about 3 nm to about 9 nm, about 5 nm to about 100 μm, about 10 nm to about 100 μm, or about 1 μm to about 50 μm.

[0076] 25. The support of any 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.

[0077] 26. The carrier of any of the preceding items, wherein at least one sample site is suitable for attachment, such as for covalent or con-covalent attachment, of a biological moiety selected from a biomolecule, a cellular part, a cellular organelle or a cell.

[0078] 27. The carrier of any of the preceding items, wherein at least one sample site is suitable for attachment, such as for covalent or co-covalent attachment of a single biomolecule.

[0079] 28. The carrier of any of the preceding items, wherein at least one selected from a biomolecule, a cell part, a cell organelle or a biological part of a cell is attached to the sample site.

[0080] 29. The carrier of any of the preceding items, wherein a single biomolecule is attached to at least one of the sample sites.

[0081] 30. The vector of any one of items 26-29, wherein the biomolecule is selected from a polypeptide, a nucleic acid, a carbohydrate and any combination thereof, wherein the biomolecule is in particular a nucleic acid polymerase, such as RNA polymerase or DNA polymerase, or a nucleic acid degrading enzyme, such as an exonuclease.

[0082] 31. The support of any of the preceding items, which is a previously used and cleaned support, which support is obtainable by subjecting a used support 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.

[0083] 32. Use of a carrier according to any one of items 1 to 31 for analyzing an event occurring at at least one sample site, wherein the event is associated with the emission of electromagnetic radiation.

[0084] 33. Use of the carrier according to any one of items 1 to 31 for analyzing single molecular events occurring at at least one sample site, wherein the single molecular event is associated with the emission of electromagnetic radiation.

[0085] 34. Use of a carrier according to any one of items 1 to 31 for individually analyzing a plurality of events, in particular single molecular events each occurring at at least one sample site, wherein the single molecular events are associated with the emission of electromagnetic radiation.

[0086] 35. The use according to item 34, wherein a plurality of events, in particular single molecular events, are analyzed in parallel.

[0087] 36. The use of any one of items 32-35, wherein the single molecular event comprises a nucleic acid sequence determination.

[0088] 37. The use according to item 36, wherein the nucleic acid sequence determination comprises:

[0089] - providing at the sample site (i) a single nucleic acid molecule, (ii) a nucleic acid synthetase molecule and / or a nucleic acid degrading enzyme molecule, and (iii) fluorescently labeled nucleotide building blocks in free form and / or incorporated into the nucleic acid molecule,

[0090] - performing an enzymatic reaction wherein nucleotide building blocks are introduced into and / or cleaved from said single nucleic acid molecule, and

[0091] - Individually determining the base sequence of nucleic acid molecules based on time-dependent fluorescence changes caused when nucleotide building blocks are introduced into and / or cleaved from individual nucleic acid molecules.

[0092] 38. The use according to item 37, wherein the nucleic acid synthetase molecule and / or the nucleic acid degrading enzyme molecule is immobilized on the sample site.

[0093] 39. Use according to any of items 32 to 38, wherein the carrier has previously been used for analysis of an event, such as a single molecule event, and has since been subjected to a cleaning procedure, wherein biological moieties, such as biomolecules, attached to at least one sample site have been removed.

[0094] 40. A method of manufacturing the carrier of any one of items 1 to 31, the method comprising:

[0095] (i) providing a substrate comprising at least one sample site on a surface of the substrate, and

[0096] (ii) coating the surface of the substrate with a layer of diamond-like carbon and / or amorphous carbon,

[0097] The material of the sample sites is selected such that it is not susceptible to the coating in step (iii), such that the layer of diamond and / or amorphous carbon does not extend over at least one sample site.

[0098] 41. A method of manufacturing the carrier of any one of items 1 to 31, the method comprising:

[0099] (i) coating the surface of the substrate with a layer of diamond-like carbon and / or amorphous carbon, and

[0100] (ii) depositing at least one sample site on the surface of the layer.

[0101] 42. A method of manufacturing the carrier of any one of items 1 to 31, the method comprising:

[0102] (i) coating the surface of a substrate with a layer of diamond-like carbon and / or amorphous carbon,

[0103] (ii) removing the layer in selected areas of the surface, and

[0104] (iii) depositing at least one sample spot in the selected area from which the layer has been removed.

[0105] 43. A method for analyzing an event, such as a single molecule event, comprising:

[0106] (ii) providing a vector according to any one of items 1 to 31,

[0107] (iii) immobilizing a biological moiety, in particular a single biomolecule, on at least one sample site of the carrier, and

[0108] (iv) analyzing events associated with said biological moiety, in particular single molecule events associated with said single biomolecule, by detecting electromagnetic radiation from said sample site.

[0109] 44. The method of claim 43, wherein the single molecular event comprises sequence analysis of a single nucleic acid molecule.

[0110] 45. An apparatus for analyzing a single molecular event, said apparatus comprising:

[0111] (i) a vector according to any one of items 1 to 31,

[0112] (ii) means for irradiating at least one sample site on the carrier with radiation, and

[0113] (iii) means for analyzing single molecular events at said at least one sample site by detecting electromagnetic radiation from said sample site.

[0114] 46. The device of claim 45, wherein the device is suitable for sequence analysis of single nucleic acid molecules.

[0115] 47. A method of cleaning a previously used support, the method 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 (d) optionally exposure to a fluorine-containing plasma.

[0116] 48. The method of item 47, comprising:

[0117] (i) providing a substrate and at least one sample site on a surface of the carrier, wherein the biological moiety is attached to the at least one sample site;

[0118] (ii) subjecting the support from step (i) to a treatment with an alkaline solution comprising 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 boiling;

[0119] (iii) optionally rinsing the support after step (ii), for example with water;

[0120] (iv) subjecting the support after step (ii) or (iii) to treatment with an acidic / oxidative solution comprising a strong mineral acid, such as sulfuric acid, and a peroxide, such as hydrogen peroxide;

[0121] (v) optionally rinsing the support after step (iv), for example, with water and / or an anhydrous organic solvent, such as ethanol;

[0122] (vi) optionally drying the support after step (iv) or (v) with an inert gas, such as Ar, N2 or any mixture thereof;

[0123] (vii) subjecting the support after step (iv), (v) or (vi) to a plasma treatment, in particular to an O2 plasma treatment and / or a fluorine-containing plasma treatment.

[0124] 49. The method of item 47 or 48, wherein the support has previously been used to analyze an event, such as a single molecule event, wherein the event is associated with the emission of electromagnetic radiation.

[0125] 50. The method of any of items 47-49, wherein the carrier in step (i) comprises a substrate and at least one sample point on the surface of the carrier, 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 point.

[0126] 51. The method of any of items 47-50, wherein the carrier in step (i) comprises a substrate and at least one sample point on the surface of the carrier, 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 sample point.

[0127] 52. The method of any of items 47-51, wherein the carrier comprises a substrate and at least one sample site on a surface of the carrier, wherein the substrate is at least partially coated with a layer of organic passivation material, and wherein the layer of organic passivation material does not extend over the at least one sample site.

[0128] 53. The method of any one of items 47-52, comprising attaching a biological moiety, such as a biomolecule, to the cleaned support after step (vii).

[0129] 54. The method of item 53, comprising optionally passivating the substrate and attaching a biomolecule to at least one sample site, wherein the support is ready for use in a new analysis of an event, such as a single molecule event.

[0130] 55. The method of item 53 or 54, comprising:

[0131] (viii) activating the surface of at least one sample site, for example, by subjecting the support after step (vii) to treatment with a thiol reagent;

[0132] (ix) optionally washing the support after step (viii), for example, with an organic solvent such as anhydrous ethanol;

[0133] (x) optionally drying the support after step (viii) or (ix) with an inert gas, such as Ar, N2 or any mixture thereof;

[0134] (xi) optionally regenerating the surface of the support, e.g., passivating the substrate, e.g., by treatment with a PEGylating agent, such as an alkoxyPEG silane, e.g., a methoxyPEG silane, or by coating the surface of the support with a layer of diamond-like and / or amorphous carbon;

[0135] (xii) optionally rinsing the support after step (xi), for example, with an organic solvent such as acetone and / or anhydrous ethanol;

[0136] (xiii) optionally drying the support after step (xi) or (xii) with an inert gas, such as Ar, N2 or any mixture thereof; and

[0137] (xiv) Binding a biomolecule to the activated surface of at least one sample spot, wherein the support is ready for use in a new assay for a single molecule event. DETAILED DESCRIPTION

[0138] 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 a "carbon film"). In certain embodiments, the support is a substantially planar support, i.e., it does not include protrusions or depressions greater than about 1000 nm or greater than about 100 nm. In other embodiments, the support is a structured support, for example, a support comprising depressions, such as a support having a diameter of about 5×10 -24 Increased to 1×10 -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 a sample point present on the carrier.

[0139] 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.

[0140] 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.

[0141] Typically, the substrate has a thickness of about 10 μm to about 5 mm, in particular about 20 μm to about 2 mm.

[0142] According to the present disclosure, the substrate is coated with a layer of a carbon film. In certain embodiments, the carbon film forms a continuous layer on the surface except in the area of the at least one sample site.

[0143] 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 a volatile gas comprising a precursor of the desired deposition material. The precursor is chemically decomposed (typically at elevated temperatures, although there are many variations of CVD 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 ranging from a few nanometers to micrometers in thickness are achievable. In ALD, the film is deposited one atomic layer at a time. The deposition process involves first exposing the surface to a linker molecule that forms a bond with the surface. A "starting material" such as water is introduced to make 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 for CVD. It combines with the linker molecule so that a single atomic layer is formed in a self-limiting manner. Thus, the film is constructed by the above-mentioned repetitive sequence. Due to the chemical interactions involved in the process, ALD is more limited than CVD in terms of the materials that can be deposited in this manner, and it can be deposited in a material-selective manner. It is also best suited for forming films with thicknesses ranging from a few nanometers to tens of nanometers.

[0144] In certain 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.

[0145] In certain embodiments, carbon films, including fluorinated carbon films, have 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.

[0146] Surfaces are described herein that are region selectively coated such that they contain a surface of a carbon film as a protein resistant region and at least one sample site suitable for attachment of a biological moiety, such as a biomolecule. In certain embodiments, the support comprises a plurality of sample sites, such as at least 10, at least 100, at least 1,000, at least 10,000 sample sites, at least 100,000, at least 1,000,000 or more sample sites. In certain embodiments, the support may comprise up to 10 6 or 10 9 or even more sample points.

[0147] In certain embodiments, the sample point includes at least one conductive material or is composed of at least one conductive material, such as a metal comprising a single metal or a combination of metals, such as an alloy or mixture of multiple different metals. For example, a metal capable of being connected to a sulfur-containing portion (e.g., in the form of a thiol or disulfide), or a metal capable of being connected to a chelating portion (e.g., a polyhistidine tag) is suitable. In certain 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 comprising at least two of the metals.

[0148] In certain embodiments, the sample site comprises or consists of at least one metal oxide, comprising a single metal oxide or a combination of metal oxides. For example, metal oxides capable of being attached to a phosphorus-containing moiety (e.g., in the form of a phosphonic acid or phosphonate), or metal oxides capable of being attached to a chelating moiety (e.g., a polyhistidine tag) are suitable. Specific examples of suitable metal oxides include TiO2 and NiO.

[0149] In further embodiments, the sample site comprises or consists of at least one non-conductive material.

[0150] The sample spots can be prepared by vapor deposition of a metal, which is vaporized on 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 point-to-point deposition of nanoparticles (e.g., having a size of 2-10 nm) by precise pipetting of the particles on the support, in particular on a support with a planar surface.

[0151] The sample spot can have a size suitable for attachment to a biological part, such as a biomolecule, a cell part, a cell organelle, or a cell, e.g., a prokaryotic or eukaryotic cell. In certain embodiments, the sample spot has a diameter of about 1 nm to about 100 μm, particularly about 2 nm to about 50 μm.

[0152] In specific embodiments, the sample spot has a size suitable for attachment of a single biomolecule. In those embodiments, the sample spot has a diameter of about 1 nm to about 100 nm, particularly about 2 nm to about 50 nm, more particularly about 5 nm to about 20 nm.

[0153] In certain embodiments, a sample point is a separate object on the surface of a substrate. In certain embodiments, the sample point has a bottom surface proximal to the substrate and a top surface distal to the substrate, wherein the distance between the bottom surface and the top surface defines the height of the sample point. In specific embodiments, the height is from about 50 μm to about 500 nanometers, particularly from about 100 μm to about 20 nm, and more particularly from about 500 μm to about 10 nm, for example, about 2 nm.

[0154] Several options exist for the arrangement of sample sites, carbon layer, and substrate.

[0155] In some embodiments, the top surface of the sample point has a height equal to that of the surrounding carbon film. In some embodiments, the top surface of the sample point is higher than the surrounding carbon film. In some embodiments, the top surface of the sample point is lower than the surrounding carbon film. The distance between the top surface of the sample point and the carbon film can be in the range of 0.1 nm to 500 nm.

[0156] In some embodiments, the sample site extends through the carbon layer such that its bottom surface is in direct contact with the surface of the substrate. In some embodiments, the sample site does not extend through the carbon layer such that its bottom surface is in direct contact with the surface of the carbon layer. In some embodiments, the sample site is located on a post that has been etched into a planar substrate, wherein a layer of diamond-like carbon and / or amorphous carbon coats the sidewalls of the post and the planar surface of the substrate.

[0157] The sample site is suitable for attaching a biological moiety, such as a biomolecule, a cellular moiety, a cellular organelle, or a cell, such as a prokaryotic or eukaryotic cell. In certain embodiments, the sample site is suitable for attaching a single biomolecule, such as by covalent or non-covalent attachment. The biomolecule can be selected from a polypeptide, a nucleic acid, a carbohydrate, and any combination thereof. In certain embodiments, the biomolecule is a nucleic acid polymerase, such as an RNA polymerase or a DNA polymerase, or a nucleic acid degrading enzyme, such as an exonuclease.

[0158] In certain embodiments, biological moieties, such as biomolecules, are directly attached to the sample site, for example, by providing a biological moiety, such as a biomolecule, with an anchoring group suitable for covalent or non-covalent attachment to the sample site, particularly a sulfur-containing group, such as a thiol group -SH, a substituted thiol group -SR, wherein R is an organic residue, such as a C1-C4 alkyl group or a disulfide group -SS-, or a phosphorus-containing group. Yet another alternative is immobilization, in which the binding of a biological moiety, such as a biomolecule, to a silica surface can be mediated via a reactive silane group.

[0159] In certain embodiments, a biological moiety, such as a biomolecule, is indirectly attached to a sample site, for example, by non-covalent high affinity attachment to a coating on the surface of the sample site. In those embodiments, a biological moiety, such as a biomolecule, can be provided having a reactive tag, such as biotin, a hapten, a poly(histidine) tag, or a carbohydrate group, capable of forming a high affinity attachment to a complementary reactive moiety, such as streptavidin, an antibody, a lectin, etc., attached to the surface of the sample site.

[0160] In certain embodiments, the fixation of the biological moiety to the support can include a coupling reaction between two bioorthogonal reactive groups, i.e., groups that are not present in the biomolecule to be attached to the sample site. In certain embodiments, the coupling reaction is a click reaction, such as a reaction between an azide group and an alkyne group, such as a terminal alkyne group or a strained alkyne group, such as a cyclooctyne group. In certain embodiments, the reaction product of the coupling reaction includes a triazole group.

[0161] In certain embodiments, a biological moiety, such as a biomolecule, can be provided that has a bioorthogonal group, such as an azide group or an alkyne group, capable of forming a covalent linkage with a complementary bioorthogonal group on a surface attached to a sample spot.

[0162] The vectors as disclosed herein can be manufactured by different procedures as outlined below.

[0163] In certain embodiments, the manufacture of the vector comprises:

[0164] (i) providing a substrate comprising at least one sample site on a surface of the substrate, and

[0165] (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),

[0166] The material of the sample sites is selected such that it is not susceptible to the coating in step (iii), such that the layer of diamond and / or amorphous carbon does not extend over at least one sample site.

[0167] According to this embodiment, examples of suitable materials for the sample sites include, but are not limited to, Au, Cu, and Ni as described in [7] and

[11] .

[0168] In certain embodiments, the manufacture of the vector comprises:

[0169] (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

[0170] (ii) depositing at least one sample site on the surface of the layer.

[0171] The vectors of the present disclosure are suitable for analyzing events occurring at a sample site, wherein the event is associated with the emission of electromagnetic radiation from the sample site. In specific embodiments, the event comprises a reaction of a biological moiety that is associated with the emission of characteristic electromagnetic radiation. In specific embodiments, the event is a single molecule event.

[0172] In specific embodiments, the vectors of the present disclosure are suitable for analyzing events, such as single molecular events occurring at at least one sample site, particularly for individually analyzing multiple single molecular events that each occur at at least one sample site, and even more particularly for individually analyzing multiple single molecular events analyzed in parallel. In specific embodiments, the single molecular event comprises a nucleic acid sequence determination.

[0173] Another embodiment relates to a method for analyzing an event, the method comprising:

[0174] (iii) providing the above-mentioned vector,

[0175] (iv) immobilizing a biological moiety, in particular a biomolecule, on at least one sample site of the carrier, and

[0176] (v) analyzing events associated with said biological moiety, in particular events associated with said biomolecule, by detecting electromagnetic radiation from said sample site.

[0177] In specific embodiments, the event is a single molecular event, and the biomolecule is a single biomolecule.

[0178] In specific embodiments, a single molecular event comprises sequence analysis of a single nucleic acid molecule.

[0179] Another embodiment relates to an apparatus for analyzing an event, the apparatus comprising:

[0180] (iv) the above-mentioned carrier, which is suitable for immobilizing biological moieties, in particular biomolecules, on at least one sample site of the carrier,

[0181] (v) means for irradiating at least one sample site of the carrier with radiation, and

[0182] (vi) means for analyzing events at said at least one sample site by detecting electromagnetic radiation from said site.

[0183] In specific embodiments, the event is a single molecular event, and the biomolecule is a single biomolecule.

[0184] In particular embodiments, the device is suitable for sequence analysis of single nucleic acid molecules.

[0185] 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.

[0186] For the analysis of single molecular events, the biomolecules are 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 1000, or at least 10,000 and at most more than 10 can be analyzed on a single support, for example a single planar support. 6 molecules.

[0187] The nucleic acid molecules to be sequenced can be selected, for example, from DNA molecules, such as genomic DNA fragments, cDNA molecules, plasmids, etc., or from RNA molecules, such as mRNA molecules. The nucleic acid molecules can be derived from a genome or an expression library, produced by a cell or organism, such as 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 at most 100,000, 10 6 or 10 7 Parallel sequencing of one or even more different nucleic acid molecules.

[0188] The nucleic acid molecules to be sequenced can be in linear or circular form, for example, single-stranded nucleic acid molecules in a covalently linked circular form. In order to obtain circular nucleic acid templates, linear nucleic acid molecules can be subjected to a cycling procedure and an optional chain separation procedure during sample preparation. Cycling can be achieved by connection according to known protocols, for example, using DNA or RNA ligase. In some embodiments, adapters and / or identifier molecules, i.e., nucleic acid molecules of known sequence, can be coupled to the nucleic acid molecules.

[0189] Sequence determination may include nucleic acid extension and / or nucleic acid degradation. The sequencing process includes one or more sequencing cycles.

[0190] Nucleic acid synthetase molecules are capable of extending a primer annealed to a nucleic acid template molecule. Primer extension can be performed by gradually introducing single nucleotide building blocks at 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 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.

[0191] Specific examples of suitable DNA polymerases include Taq polymerase, exonuclease-deficient Taq polymerase, E. coli DNA polymerase I, Klenow fragment, reverse transcriptase, Φ29-related polymerase (including wild-type Φ29 polymerase) and derivatives of such polymerases such as exonuclease-deficient, T7 DNA polymerase, T5 DNA polymerase, RB 69 polymerase, etc.

[0192] Nucleic acid degrading enzyme molecules can progressively cut individual nucleotide building blocks from nucleic acid molecules. Preferably, exonucleases are used, more preferably single-stranded exonucleases that degrade along a 3' → 5' direction or along a 5' → 3' direction. Particularly preferred exonucleases are 3' → 5' exonucleases (such as E. coli exonucleases I and E. coli exonucleases III) and 5' → 3' exonucleases (such as T7 exonucleases, E. coli exonucleases II and E. coli exonucleases VIII). In addition, various polymerases can be used, such as the exonuclease activity of Klenow fragments, Taq polymerases, or T4 polymerases.

[0193] Nucleic acid synthetase molecule and linear or annular nucleic acid template molecule, for example single-stranded DNA or RNA molecule and with nucleic acid template molecule annealing or can contact with its annealed primer molecule.Primer molecule is preferably single-stranded nucleic acid or nucleic acid analog molecule with free 3 ' end, and this free 3 ' end can be extended 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, and for example maximum 20, 25, 50 or 100 nucleotide, or even higher.In some embodiments, the digestion of primer resistant nucleic acid degradation enzyme molecule, for example, by introducing the connecting base between nucleotide analog building block and / or the nucleotide building block, this is stable for degraded.In other embodiments, primer is sensitive to the digestion by nucleic acid degradation enzyme molecule.

[0194] The sequence of the primer is selected because it effectively anneals with the template molecule under the reaction conditions. For example, the primer can be a universal degenerate primer (degenerated primer) that can statistically anneal with an unknown nucleic acid sequence. In other embodiments, the primer can partially anneal with the known sequence of the nucleic acid template molecule. In this embodiment, known adapters and / or identifier sequences can be introduced into the nucleic acid template molecule. The primer can be unlabeled or include a fluorescent marker group.

[0195] Furthermore, it is required that there are nucleotide building blocks carrying at least one fluorescent marker group. Preferably, each different nucleotide building block (A, G, C, T / U) contains a different fluorescent marker group.

[0196] The fluorescent labeling group can be selected from known fluorescent labeling groups used to label biopolymers, especially nucleic acids, such as fluorescein dyes, rhodamine, oxazines, such as Evoblue or Gnothis Blue, phycoerythrin, Cy3, Cy5, IR dyes or their derivatives, etc.

[0197] The nucleotide building blocks may carry (i) a fluorescent marker group that remains on the building block when it is introduced 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 it is introduced into a nucleic acid molecule during primer extension catalyzed by a nucleic acid synthetase molecule. The fluorescent marker group that remains on the building block is preferably linked to an α-phosphate group, to a sugar, and / or to a nucleobase group.

[0198] In a specific embodiment, the fluorescent marker group retained in the building block is connected to core base, for example, via connexon, and this connexon can have maximum 15, preferably 10-12 carbon atoms, optionally includes heteroatoms, for example the chain length of N, O or S atoms.When the building block is introduced into nucleic acid molecules, the fluorescent marker group that is cut can be connected to terminal phosphate group, for example polyphosphoric acid building block, includes but is not limited to the terminal phosphate group of six-, five-, four-or triphosphate building block, such as the gamma-phosphate group of triphosphate building block.In certain embodiments, building block is selected, the fluorescent marker group that the building block contains (i) retains after introducing and (ii) is cut during introducing.In this case, the fluorescent group that can interact with each other (for example, by quenching and / or energy transfer) can be selected.

[0199] 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.

[0200] The sequencing procedure can include a step of generating a nucleic acid molecule having nucleotide building blocks introduced 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 primer extension and / or during degradation.

[0201] Sequence determination during primer extension involves the use of nucleotide building blocks carrying a fluorescent marker group, which, when introduced into a nucleic acid molecule, is cut from the building block. In this case, the time-dependent fluorescence change caused by cutting the fluorescent marker group from the building block can be measured. Sequence determination during nucleic acid degradation involves the use of nucleotide building blocks, which carry a fluorescent marker group, which, when introduced into a nucleic acid molecule, is retained on the building block. When the labeled nucleotide building blocks are released from the nucleic acid molecule, the stepwise cutting of the individual nucleotide building blocks from the nucleic acid molecule causes a time-dependent change in fluorescence. In certain embodiments, sequence determination can also be performed during extension and degradation, that is, when using such nucleotide building blocks, when the building block is introduced into a nucleic acid molecule, it carries both a fluorescent marker group retained in the building block and a fluorescent marker group cut from the building block. In this embodiment, the two fluorescent groups can be the same or different.

[0202] In some embodiments, the method involves 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 involves the 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.

[0203] When the nucleotide building blocks are introduced into the extended nucleic acid molecules, a time-dependent change in fluorescence can occur, which can be detected as described above. Preferably, the nucleotide building blocks are introduced into the extended nucleic acid molecules in association with a detectable increase in fluorescence, preferably in association with an instantaneous increase in fluorescence. For example, a nucleotide building block can be used which carries a fluorescent marker group on the portion of the molecule that is cut off (e.g., on a γ-phosphate group) when the building block is introduced into a primer.

[0204] When a nucleotide building block is cleaved from a synthetic nucleic acid molecule, the fluorescent marker group introduced into the nucleic acid chain interacts with adjacent groups, for example, chemical groups of the nucleic acid, in particular a nucleobase, such as G, and / or adjacent fluorescent marker groups, and these interactions lead to changes in fluorescence, in particular in fluorescence intensity, compared to the "isolated" form of the fluorescent marker group due to quenching processes and / or energy transfer processes, and a time-dependent change in fluorescence can be measured. The removal of individual nucleotide building blocks by cleavage changes the overall fluorescence, for example, the fluorescence intensity of an immobilized nucleic acid chain, and this change is a function of the removal of the individual nucleotide building blocks by cleavage, i.e., a function of time.

[0205] 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 reverse-bound labeled nucleotides that are free to move during polymerization.

[0206] 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 the individual nucleic acid strands. Preferably, a fluorescent marker group is used which, when introduced into a nucleic acid strand, is at least partially quenched, so that the fluorescence intensity increases after the nucleotide building block containing the marker group or the adjacent building block causing the quenching has been removed by cleavage.

[0207] During the introduction and / or removal of individual nucleotide building blocks, changes in the fluorescence intensity of the nucleic acid chain and / or the introduced or cleaved nucleotide building blocks can be measured due to quenching or energy transfer processes. This change in fluorescence intensity over time depends on the base sequence of the nucleic acid chain under investigation and can therefore be correlated with the sequence.

[0208] 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 (e.g., 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 linked to the nucleic acid strand to be investigated, e.g., by an enzymatic reaction using a ligase and / or a terminal transferase, so that an initially known fluorescence pattern is obtained at the beginning of sequencing, and only thereafter a fluorescence pattern corresponding to the unknown sequence to be investigated is obtained.

[0209] Detection includes irradiating light into the carrier, preferably by means of a laser or by another suitable light source, to cause excitation of the fluorescent marker group. In this regard, one or more laser beams can be used, for example, an extended laser beam with a cross section of about 1-20 mm, and / or multiple laser beams. Detection preferably includes multi-point fluorescence excitation by a laser, for example, a dot matrix of laser points generated via diffraction optics (see WO 2002 / 097406) or a quantum well laser.

[0210] Fluorescence emission from multiple nucleic acid strands can be detected in parallel using a detector matrix, such as 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 in a manner that fluorescence excitation and detection are performed in parallel on some or all of the nucleic acid strands being studied. Preferably, detection is performed on fluorescent light emitted from the carrier surface substantially orthogonally through the reaction space or through the carrier body.

[0211] Detection can be performed, for example, by means of single molecule detection, for example by fluorescence correlation spectroscopy, which involves the integration 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, said light exciting the receptors present in the measurement volume so that the latter emit fluorescent light, the fluorescent light emitted from the measurement volume being measured by means of a light detector, and the variation of the measured emission over time being correlated with the concentration of the analyte, making it possible to identify individual molecules in the measurement volume at appropriately high dilutions. Details of the procedures and equipment for detection can be found in the disclosure of EP 0 679 251, the contents of which are incorporated herein by reference. In addition, confocal determination of single molecules is 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.

[0212] Alternatively or additionally, detection can also be performed by time-resolved decay measurement (known as "time gating"), as described, for example, by Rigler et al. in "Picosecond Single Photon Fluorescence Spectroscopy of Nucleic Acids" in "Ultrafast Phenomena", ed. D.H. Auston, Springer, 1984, the contents of which are incorporated herein by reference. In this case, fluorescent molecules are excited in the measurement volume and then the detection interval on the photodetector is switched on, for example, at time intervals of ≥100 ps. In this way, the background signal generated by the Raman effect can be kept sufficiently low so that single molecules can be detected in a substantially interference-free manner.

[0213] A second aspect of the present disclosure relates to a method for cleaning a previously used carrier, wherein the carrier is suitable for use in an apparatus and method for analyzing an event, such as a single molecule event. If not stated differently, features of the first aspect also apply to the second aspect.

[0214] As understood herein, a previously used carrier encompasses a carrier on which a previous analysis, such as a single molecule analysis, has been performed. Thus, the carrier has already attached a biological moiety, such as a biomolecule, to at least one sample site and is optionally contaminated with additional components, such as components of the sample that was analyzed. By the methods disclosed herein, the biological moiety, such as a biomolecule, and optionally the additional components attached to the sample site are removed from the carrier without causing substantial damage.

[0215] This aspect relates to the reusability of the carrier. Once the carrier has been used for its desired application, it can include a sample point, such as a metal sample point on a substrate (e.g., glass), which is surrounded by a diamond-like carbon and / or amorphous carbon (DLC or α-carbon) film, to which biomolecules or residues of biomolecules are bound together with possible residues from reagents. In some cases, biomolecule residues and reagent residues are also present in the coated area. In order to return the surface to the "original" condition necessary for their reuse, the surface must undergo a thorough cleaning to remove residues and residues.

[0216] Alkaline solutions are effective in removing nucleic acids, such as DNA or RNA deposits. They are available as commercial products (e.g., DNA Away) and can be used to treat spent carriers. Treatment can be performed at about 20°C to 25°C or at an elevated temperature, such as about 40°C to enhance effectiveness. Acidic / oxidizing solutions, such as "piranha solution" consisting of a mixture of sulfuric acid and hydrogen peroxide, are efficient in removing proteins and protein deposits. These treatments can be performed sequentially, optionally with intermediate rinsing steps. These cleaning steps should leave the surface - the metal sample point surrounded by the diamond-like and / or amorphous carbon film - essentially unaffected.

[0217] An additional cleaning step consisting of exposure to an O plasma can provide even further cleaning, however, it can remove some of the DLC or α-carbon film, so it should be performed with caution. In certain embodiments, the diamond-like carbon and / or amorphous carbon film can then be refluorinated by exposure to a fluorine-containing plasma, for example as in the original film formation.

[0218] In one embodiment of this aspect, the entire DLC or α-carbon film is removed by etching in an O plasma, substantially exposing the underlying glass substrate and (one or more) metal sample sites. The substrate can then be calcined at a high temperature to ensure that the metal sample sites present a fresh surface for biomolecule attachment. If the sample sites are nanoparticles, they can be melted and reformed in this process. The formation of a new DLC or α-carbon film is then achieved as described herein above.

[0219] Embodiments of the second aspect are directed to a method of cleaning a previously used support, the method comprising subjecting the used support to (a) treatment with an alkaline solution, (b) treatment with an acidic / oxidizing solution, optionally (c) exposure to an O plasma, and (d) optionally exposure to a fluorine-containing plasma.

[0220] A particular embodiment of the second aspect relates to a method of cleaning a previously used carrier, the method comprising:

[0221] (i) providing a carrier comprising a substrate and at least one sample site on a surface of the carrier, wherein a biological moiety, such as a biomolecule, is attached to the at least one sample site;

[0222] (ii) subjecting the support from step (i) to a treatment with an alkaline solution, including, 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 elevated temperature, in particular at boiling;

[0223] (iii) optionally rinsing the support after step (ii), for example with water;

[0224] (iv) subjecting the support after step (ii) or (iii) to treatment with an acidic / oxidative solution comprising a strong mineral acid, such as sulfuric acid, and a peroxide, such as hydrogen peroxide;

[0225] (v) optionally rinsing the support after step (iv), for example, with water and / or an anhydrous organic solvent, such as ethanol;

[0226] (vi) optionally drying the support after step (iv) or (v) with an inert gas, such as Ar, N2 or any mixture thereof;

[0227] (vii) optionally subjecting the support after step (iv), (v) or (vi) to a plasma treatment, in particular to an O2 plasma treatment.

[0228] In certain embodiments of this aspect, the carrier to be cleaned comprises a substrate and at least one sample site on a surface of the carrier, 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 at least one connection point.

[0229] In a further embodiment of this aspect, the carrier to be cleaned comprises a substrate and at least one sample point on a surface of the carrier, wherein the substrate is at least partially coated with an organic passivation material, such as a layer of a passivation material containing poly(ethyleneoxy) groups, and wherein the layer of organic passivation material does not extend over the at least one sample point.

[0230] In some embodiments of this aspect, biological part such as biomolecule is connected to the carrier through cleaning after step (vii).In addition, the carrier surface around the sample point can be regenerated, for example, with the passivation reagent treatment that suppresses biomolecule such as protein adhesion, to suppress the adhesion of biomolecule and / or other sample components.In certain embodiments, passivation substrate can include using PEGylation reagent such as alkoxy PEG silane, for example methoxy PEG silane treatment.Alternatively, regeneration can include the surface of the carrier coated with a fresh layer of diamondoid and / or amorphous carbon.After this, the carrier is ready for event, for example, new analysis of single molecule event.Typically, regeneration step is carried out before the step of connecting biological part, for example, biomolecule.When using as described above in the first aspect, the carrier coated with carbon film, in certain embodiments, regeneration step is not necessary.

[0231] In some embodiments, the attachment of a biological moiety, such as a biomolecule, comprises:

[0232] (viii) activating the surface of at least one sample site, for example, by subjecting the support after step (vii) to treatment with a thiol reagent;

[0233] (ix) optionally washing the support after step (viii), for example, with an organic solvent such as anhydrous ethanol;

[0234] (x) optionally drying the support after step (viii) or (ix) with an inert gas, such as Ar, N2 or any mixture thereof;

[0235] (xi) optionally regenerating the surface of the support, e.g., passivating the substrate, e.g., by treatment with a PEGylating agent, such as an alkoxyPEG silane, e.g., a methoxyPEG silane, or by coating the surface of the support with a layer of diamond-like and / or amorphous carbon;

[0236] (xii) optionally rinsing the support after step (xi), for example, with an organic solvent such as acetone and / or anhydrous ethanol;

[0237] (xiii) optionally drying the support after step (xi) or (xii) with an inert gas, such as Ar, N2 or any mixture thereof; and

[0238] (xiv) Binding biomolecules to the activated surface of at least one sample spot, wherein the support is ready for new analysis of single molecule events.

[0239] Furthermore, the present disclosure is explained in detail by referring to the following specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0240] Figure 1 Embodiment 1 (Prior Art) of the present invention 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.

[0241] Figure 2 Embodiment 2 (Prior Art) of the present invention shows an optically transparent substrate (e.g., quartz; fused silica; or glass) coated with a layer of (DLC) or amorphous carbon, deposited by atomic layer deposition (ALD) with a thickness ranging from about 1 nm to 100 nm. After deposition, the film is exposed to a fluorine-containing plasma.

[0242] Figure 3A and 3BEmbodiments 3 and 4 of the present disclosure show an optically transparent substrate (e.g., quartz; fused silica; or glass) coated with a DLC deposited by CVD or ALD to a thickness ranging from 1 nm to 10 μm. After deposition, the DLC is coated with a photoresist, optionally after treatment with an oxygen plasma. The photoresist is then patterned by photolithography (e.g., optical or electron beam lithography, or any other suitable technique) and developed according to the desired pattern to form a template in the resist.

[0243] according to Figure 3A , there is an underlying metal or metal oxide layer. Openings are formed in the carbon film by etching to expose selected areas of the underlying film. These openings can form sample sites. The carbon film is exposed to a fluorine-containing plasma.

[0244] according to Figure 3B , creating metal or metal oxide features on top of the carbon film, for example by conventional liftoff patterning.The carbon film is exposed to a fluorine-containing plasma.

[0245] Figure 4A and 4B Embodiment 5 of the present disclosure shows an optically transparent substrate (e.g., quartz; fused silica; or glass) that is patterned with metal dots using a photolithographic patterning method such that the pattern of metal dots is arranged at predetermined locations on the surface of the substrate. The metal is a non-adhesive material, that is, it does not react with DLC (or in the case of ALD, with ALD linkers). Au, Cu, and Ni are examples of such metals [7]. The surface is then selectively coated with DLC. Because DLC does not bind to the metal, carbon will not be deposited on the metal, but only on the glass. The support is exposed to a fluorine-containing plasma.

[0246] Embodiment 6 (of the present disclosure) comprises a solid substrate (e.g., glass) and at least one sample site or a plurality of sample sites on a surface of the substrate, wherein the sample site is designed to accommodate a single biomolecule of interest, or at most a small number of biomolecules, e.g., up to 5 biomolecules of interest

[15] . In specific embodiments, the sample sites have lateral and longitudinal dimensions within a size range of less than 10 nm, but not more than 20 nm, e.g., from about 2 nm to about 20 nm, or from about 5 nm to about 20 nm. The sample sites can be formed using direct photolithographic techniques to form thin metal platelets composed of the desired metal material

[16] , or they can take the form of nanoparticles produced using a combination of photolithographic patterning and post-processing that produces spherical or spherical metal particles of the desired size and shape [15, 17-19].

[0247] In this embodiment, the sample points are organized in a predetermined spatial arrangement on the substrate so that the position of each single biomolecule at the sample point is known. This facilitates subsequent detection of the interaction of the (one or more) biomolecules of interest by a detector system (e.g., fluorescence). The spatial arrangement can be an ordered pattern, such as according to a Cartesian grid, a hexagonal array, a Fibonacci diagram / layout, etc., with a distance between points ranging from 20 nm to more than 10 μm, so that the position of each sample point on the substrate is known, within an accuracy range of about + / - 5 nm, + / - 10 nm, or + / - 25 nm. Such an arrangement can be provided by nanolithographic patterning techniques (e.g., electron beam lithography, nanoimprint lithography, extreme ultraviolet lithography, etc.) and optional additional processing.

[0248] The sample points in this embodiment are composed of metal and can be manufactured by methods such as those described in references [15, 17-19] or any other photolithographic [15-19] or photolithographically directed self-assembly method that can produce metallic features in the sub-20 nm size range. Attachment of the biomolecule of interest is achieved by one of several techniques described in the literature (see, for example, references [15, 17-19]).

[0249] To ensure that the biomolecule of interest is confined to the sample site and not bound or adsorbed by 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 to a thickness less than the height of the sample site, which remains exposed for further reaction with the selected biomolecule(s). In certain embodiments, the layer thickness is at least about 1 nm and at most about 0.1 nm less than the height of the sample site, for example, for a sample site height of 5 nm, the layer thickness is 1 to 4.9 nm, or for a sample site height of 20 nm, the layer thickness is 1 to 19.9 nm.

[0250] The DLC or α-carbon film can be fluorinated in situ during deposition or post-deposition (eg, by exposure to a fluorine-containing plasma).

[0251] This selective deposition can be accomplished by either of two means:

[0252] (i) DLC or α-carbon films are grown selectively on glass and not on sample site materials (e.g., gold) by the choice of deposition conditions and / or deposition precursors. This approach is particularly relevant to atomic layer deposition (ALD), where the precursor [20, 21] can be chosen so that it selectively binds to the glass substrate and not to the sample site, ensuring that the DLC or α-carbon film [22, 23] grows only on the area surrounding the sample site, e.g., Figure 5 As in a and b.

[0253] (ii) Alternatively or additionally, the site may be protected by, for example, a self-assembled monolayer (SAM) attached only to the sample site

[21] . The SAM prevents the growth of DLC or α-carbon films on the site, e.g. Figure 5 ce.

[0254] Implementation Plan 7 (The present disclosure) follows the basic layout of embodiment 6, except for the method of forming the DLC or α-carbon film around the sample point. In this embodiment, the DLC or α-carbon film is deposited on the entire surface to a thickness large enough to cover the sample point, such as Figure 6 The deposited film is then etched back, for example by reactive ion etching in oxygen plasma, to expose the top of the sample site, while the DLC or α-carbon film of reduced thickness remains around the sample site, as in Figure 6 As in c.

[0255] For large area spots and relatively thick deposited films, this method can result in complete removal of the film outside the sample spot due to the small or (possibly) zero difference in thickness at and outside the sample spot. For nanometer-sized spots and relatively thin deposited films, e.g., films having a thickness of about 100 nm or less, particularly about 20 nm or less, the etching method can be precisely calibrated to expose the top of the sample spot without removing the deposited film outside those areas ( Figure 6 c). The DLC or α-carbon film can be fluorinated in situ during deposition or post-deposition (eg by exposure to a fluorine-containing plasma).

[0256] References:

[0257] 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.

[0258] 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.

[0259] 3.Park,S.R.,et al.,A Single-Molecule Surface-Based Platform to Detectthe Assembly and Function ofthe Human RNA Polymerase II TranscriptionMachinery.Structure,2020.28(12):p.1337-1343.e4.

[0260] 4.Wong,I.and C.-M.Ho,Surface molecular property modifications forpoly(dimethylsiloxane)(PDMS)based microfluidic devices.Microfluidics andNanofluidics,2009.7(3):p.291.

[0261] 5.Yonezawa,K.,et al.,Evaluation of Antibacterial and CytotoxicProperties of a Fluorinated Diamond-Like Carbon Coating for the Developmentof Antibacterial Medical Implants.Antibiotics(Basel),2020.9(8).

[0262] 6.Horikawa,A.M.,Shunto;Hasebe,Terumitsu;Matsumoto,Tomohiro;Tanaka,Minoru;akahashi,Koki;Suzuki,Tetsuya,Fluorine-incorporated amorphous carboncoating inhibits adhesion of blood cells to biomaterials.Sensors andMaterials,2017.29(6):p.9.

[0263] 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 hardcoatings,in Sandia Report.1994.

[0264] 8.Rajak,D.K.,et al.,Diamond-Like Carbon(DLC)Coatings:Classification,Properties,and Applications.Applied Sciences,2021.11(10):p.4445.

[0265] 9.Robertson,J.,Diamond-like amorphous carbon.Materials Science andEngineering:R:Reports,2002.37(4):p.129-281.

[0266] 10.Bendavid,A.,et al.,The properties of fluorine-containing diamond-like carbon films prepared by pulsed DC plasma-activated chemical vapourdeposition.Diamond and Related Materials,2010.19(12):p.1466-1471.

[0267] 11.Schvartzman,M.,et al.,Plasma fluorination of carbon-basedmaterials for imprint and molding lithographic applications.Applied PhysicsLetters,2008.93(15):p.3.

[0268] 12.Schvartzman,M.,et al.,Fluorinated diamondlike carbon templates forhigh resolution nanoimprint lithography.Journal of Vacuum Science&TechnologyB,2008.26(6):p.2394-2398.

[0269] 13.Carvalho,I.,et al.,Overview on the Antimicrobial Activity andBiocompatibility of Sputtered Carbon-Based Coatings.Processes,2021.9(8):p.1428.

[0270] 14.Hasebe,T.,et al.,Fluorine doping into diamond-like carbon coatingsinhibits protein adsorption and platelet activation.Journal of BiomedicalMaterials Research Part A,2007.83A(4):p.1192-1199.

[0271] 15.Cai,H.,et al.,Molecular Occupancy of Nanodot Arrays.ACS Nano,2016.10(4):p.4173-4183.

[0272] 16.Cherniavskaya,O.,et al.,Fabrication and surface chemistry ofnanoscale bioarrays designed for the study of cytoskeletal protein bindinginteractions and their effect on cell motility.Journal of Vacuum Science&Technology B,2005.23(6):p.2972-2978.

[0273] 17.Cai,H.,et al.,Full control of ligand positioning reveals spatialthresholds for T cell receptor triggering.Nature Nanotechnology,2018.13(7):p.610-617.

[0274] 18.Penzo,E.,et al.,Directed Assembly of End-Functionalized SingleWall Carbon Nanotube Segments.Nano Letters,2015.15(10):p.6547-6552.

[0275] 19.Schvartzman,M.,et al.,Nanolithographic Control of the SpatialOrganization of Cellular Adhesion Receptors at the Single-Molecule Level.NanoLetters,2011.11(3):p.1306-1312.

[0276] 20.Cao,K.,J.Cai,and R.Chen,Inherently Selective Atomic LayerDeposition and Applications.Chemistry of Materials,2020.32(6):p.2195-2207.

[0277] 21.Oh,I.-K.,et al.,Role of Precursor Choice on Area-Selective AtomicLayer Deposition.Chemistry of Materials,2021.33(11):p.3926-3935.

[0278] 22.Choi,T.,et al.,Hydrogen plasma-enhanced atomic layer deposition ofhydrogenated amorphous carbon thin films.Surface and Coatings Technology,2018.344:p.12-20.

[0279] 23.Xiao,Z.,K.Kisslinger,and R.Monikandan,Atomic Layer Deposition ofNanolayered Carbon Films.C,2021.7(4):p.67.

Claims

1. A carrier comprising a substrate and at least one sample site on a surface of the carrier, 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 binding site.

2. The carrier according to claim 1, which is at least substantially planar.

3. A carrier according to any one of the preceding claims, comprising a plurality of sample sites.

4. The carrier according to any one of the preceding claims, wherein the at least one sample site comprises at least one metal or metal oxide. 5 . The carrier according to claim 1 , wherein the at least one sample spot has a diameter of about 1 nm to about 100 μm, in particular about 2 nm to about 50 nm, and more in particular about 5 nm to about 20 nm.

6. A carrier according to any one of the preceding claims, (i) wherein the at least one sample site has a top surface remote from the substrate, and wherein the top surface has a height equal to that of a surrounding layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon; (ii) wherein the at least one sample site has a top surface remote from the substrate, and wherein the top surface of the sample site is higher than a surrounding layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon; or (iii) wherein the at least one sample site has a top surface remote from the substrate, and wherein the top surface of the sample site is lower than the surrounding layer of fluorinated diamond-like carbon and / or fluorinated amorphous carbon.

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

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

9. The carrier according to any 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 polymerase, such as an RNA polymerase or a DNA polymerase.

10. The carrier according to any one of the preceding claims, which is a previously used and cleaned carrier, obtainable by subjecting a used carrier to: (a) treatment with an alkaline solution, (b) treatment with an acidic / oxidizing solution, optionally (c) exposure to an O plasma, and d) optionally exposure to a fluorine-containing plasma.

11. Use of a carrier 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 the sample site, in particular a single molecule event comprising nucleic acid sequence determination, the event occurring at the at least one sample site, in particular for individually analyzing a plurality of single molecule events each occurring at the at least one sample site, and more particularly for individually analyzing a plurality of single molecule events analyzed in parallel.

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

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

14. An apparatus for analyzing an event, the apparatus comprising: (i) the vector according to any one of claims 1 to 10, (ii) means for irradiating said at least one sample site on said carrier with radiation, and (iii) means for analyzing events at said at least one sample site by detecting electromagnetic radiation from said site.

15. The device according to claim 14, which is suitable for sequence analysis of single nucleic acid molecules.

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