Observation sample and kit

Abstract

This technology provides a method for analyzing the minute dynamics of self-assembled molecular films or biomolecules in a near-natural state. [Solution] An observation sample (100) for analyzing the dynamics of biomolecules (13), characterized by comprising: a substrate (11) having a diamond crystal layer (111) on which NV centers (111c) are arranged; a molecular film (12) formed along the surface of the substrate and composed of self-assembled molecules; and biomolecules trapped in the molecular film.

Description

Technical Field

[0001] The present disclosure relates to an observation sample and a kit.

Background Art

[0002] In recent years, techniques for analyzing the dynamics of various molecules present in living organisms using quantum sensors have been developed.

[0003] As an example, Non-Patent Document 1 discloses a technique for analyzing the dynamics of a lipid bilayer formed on the diamond surface by nano-scale NMR using a nitrogen-vacancy (NV) center in diamond as a quantum sensor. Further, Non-Patent Document 2 discloses a technique for mapping paramagnetic species in Gd 3+ -containing liposomes and control liposomes by electron spin resonance (ESR) spectroscopy. Further, Patent Document 1 discloses a microchamber including a diamond substrate provided with a recess and a diamond crystal layer formed on the side surface of the recess and having an NV center, which can be used for bioassay.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0006] Incidentally, cryo-electron microscopy and X-ray crystallography have been used to analyze the dynamics of biomolecules trapped in self-assembled molecular membranes, such as membrane proteins in lipid bilayers. While cryo-electron microscopy and X-ray crystallography exhibit high spatial resolution, they require extremely low temperatures of approximately -70°C, meaning that the dynamics of self-assembled molecular membranes or biomolecules are sometimes analyzed under conditions that differ significantly from their natural state, such as in vivo.

[0007] While the technologies disclosed in Non-Patent Documents 1 and 2, and Patent Document 1, are applicable to the analysis of specific molecules, further development of technologies for analyzing the minute dynamics of self-assembled molecular films or biomolecules in near-natural conditions is desired.

[0008] One aspect of this disclosure aims to provide a technique for analyzing the minute dynamics of self-assembled molecular films or biomolecules in a near-natural state. [Means for solving the problem]

[0009] To solve the above problems, an observation sample according to one aspect of the present disclosure is an observation sample for analyzing the dynamics of biomolecules, comprising: a substrate having a diamond crystal layer on which NV centers are arranged; a molecular film formed along the surface of the substrate and composed of self-assembled molecules; and the biomolecules trapped in the molecular film.

[0010] To solve the above problems, an observation sample according to one aspect of the present disclosure is an observation sample for dynamic analysis of a molecular film, comprising: a substrate having a diamond crystal layer on which NV centers are arranged; and a molecular film formed along the surface of the substrate and composed of self-assembled molecules, wherein the molecular film is formed in an island-like manner on the surface of the substrate and comprises at least one island-like region on the surface of the substrate and a sea-like region surrounding the at least one island-like region on the surface of the substrate where the molecular film is not formed.

Advantages of the Invention

[0011] According to one aspect of the present disclosure, a technique for analyzing the minute dynamics of self-assembled molecular films or biomolecules in a state close to nature is provided.

Brief Description of the Drawings

[0012] [Figure 1] It is a schematic diagram showing the configuration of an observation sample according to Embodiment 1 of the present disclosure. [Figure 2] It is a schematic diagram showing a method for preparing the observation sample shown in FIG. 1. [Figure 3] It is a schematic diagram showing the configuration of an observation sample according to Embodiment 2 of the present disclosure. [Figure 4] It is a fluorescence microscope image of the observation sample of Example 1 of the present disclosure. [Figure 5] It is a microscope image of the observation sample of Example 1 of the present disclosure and the result of quantum measurement imaging. [Figure 6] It is another fluorescence microscope image of the observation sample of Example 1 of the present disclosure. [Figure 7] It is an AFM microscope image of the observation sample of Example 1 of the present disclosure.

Modes for Carrying Out the Invention

[0013] 〔Observation Sample〕 The observation sample according to one aspect of the present disclosure will be described below. The observation sample according to one aspect of the present disclosure has a substrate having a diamond crystal layer in which NV centers are arranged, and a molecular film composed of self-assembled molecules arranged along the surface of the substrate.

[0014] (Substrate)​​​​​The NV (nitrogen-vacancy) center is a composite defect in a diamond crystal where nitrogen substitutes for carbon that should originally be present, and there is a vacancy at an adjacent position. The substrate having a diamond crystal layer may be manufactured by known techniques. As an example of the manufacturing method of the substrate: the CVD method using a nitrogen-containing gas; the ion implantation method using nitrogen ions; the electron beam irradiation method of irradiating diamond previously added with nitrogen with an electron beam; etc. can be mentioned. As a specific example of the manufacturing method, a diamond plate having no NV center and having a crystal plane oriented to

[0111] , and using a mixed gas containing carbon and nitrogen as a raw material, bringing the plate into contact with the mixed gas using a technique such as CVD, and growing diamond crystals on the plate can be mentioned.

[0016] The diamond crystal layer may be planar or may have any irregularities. As an example, the diamond crystal layer may have a concave portion. Such a concave portion may be formed by growing diamond crystals on a diamond plate having a concave portion as well.

[0017] In the diamond crystal layer, a plurality of NV centers are arranged along the first surface near the surface (hereinafter sometimes referred to as "the first surface of the substrate") that forms a molecular film among the two surfaces of the substrate.

[0018] The detection region by each NV center spreads concentrically from the NV center and has a volume of about 6 nm. 3 When the NV centers are sparsely arranged so that the detection regions by each NV center do not overlap with each other, the plurality of NV centers show responsiveness to molecules transported in a direction parallel or perpendicular to the first surface as a whole. Therefore, the plurality of NV centers can detect the molecular transport in the parallel and perpendicular directions of the molecules.

[0019] From the viewpoint of further improving the detection sensitivity by the NV center, the depth from the first surface of the substrate to the NV center is preferably as small as possible, and as an example, it is 10 nm or less.

[0020] From the viewpoint of further improving the detection sensitivity by the NV center, it is preferable that the substrate has a diamond crystal layer as the outermost layer on the first surface side of the substrate. However, as long as the detection area by the NV center includes the first surface of the substrate, the diamond crystal layer does not have to be the outermost layer on the first surface side of the substrate. In other words, the substrate may have other layers on the first surface side of the diamond crystal layer.

[0021] Other examples of layers include surface treatment layers for adjusting the properties of the first surface of the substrate, such as hydrophilicity or charge. Surface treatment to adjust the properties of the first surface of the substrate promotes the formation of the molecular film, described later, along the first surface of the substrate. In other words, it is preferable to adjust the properties of the first surface of the substrate according to the type of molecules that make up the molecular film. Examples of surface treatments include acid treatment or base treatment to improve the hydrophilicity of the first surface.

[0022] (molecular membrane) A molecular film is composed of self-assembled molecules formed along a first surface of a substrate. In one embodiment of this disclosure, the molecular film can capture biomolecules that are the subject of dynamic analysis and can interact with such biomolecules in various forms, such as chemical reactions or molecular transport. Alternatively, in one embodiment of this disclosure, the molecular film may be the subject of dynamic analysis. Hereinafter, molecules that constitute a molecular film by self-assembly may be referred to as "membrane molecules."

[0023] The molecular film is formed along the first surface of the substrate. In such molecular films, the distance from the membrane molecule to the NV center varies less from molecule to molecule compared to lipid membrane mimics such as micelles, liposomes, and bicelles simply deposited on the substrate. Therefore, the variation in the signal intensity detected by the NV center for membrane molecules and the biomolecules captured by the membrane molecules is also small, enabling highly accurate dynamic analysis of biomolecules and molecular films.

[0024] Molecular films are composed of self-organized molecules. While the molecular film as a whole maintains a film-like structure, the membrane molecules that make up the film exhibit fluidity and can move within the film by diffusion or by anisotropic or isotropic transport.

[0025] Film molecules move within the molecular film. Therefore, controlling the area of ​​the molecular film limits the range of movement of film molecules parallel to the first surface of the substrate to the controlled area. This limitation is advantageous from the viewpoint of quantitatively evaluating the movement distance and direction of film molecules compared to a case where the area of ​​the molecular film is not controlled. One example of a method for controlling the area of ​​the molecular film is to form the molecular film only on a specific part of the first surface of the substrate, rather than the entire surface. Another preferred example of a method for controlling the area of ​​the molecular film is to form the molecular film on the first surface of the substrate in a sea-island structure. In other words, it is preferable that the observation sample has at least one island-like region on the first surface of the substrate where the molecular film is formed in an island-like manner, and a sea-like region surrounding at least one island-like region where no molecular film is formed. Furthermore, from the viewpoint of improving throughput, it is even more preferable that the observation sample has multiple island-like regions separated by sea-like regions.

[0026] When the island-like regions are circular, the diameter of the island-like regions is preferably 1 μm to 100 μm, from the viewpoint of performing a more quantitative analysis of the dynamics of the molecular film. Controlling the diameter of the island-like regions in this way is also advantageous from the viewpoint of controlling the area of ​​the island-like regions to be small, thereby controlling the number of biomolecules per molecular film to an extremely small amount and enabling single-molecule analysis of biomolecules. When the island-like regions are not circular, it is preferable to adjust the dimensions of the island-like regions so that they have the same area as a circle with a diameter within the above range.

[0027] The spacing between island-like regions, i.e., the width of the sea-like regions, is preferably 1 μm to 100 μm from the viewpoint of preventing linkage between adjacent island-like regions due to the dispersion of membrane molecules while improving throughput.

[0028] In this disclosure, the molecular film may be formed over the entire first surface of the substrate, from the viewpoint of simplifying the preparation of the observation sample. Even when the molecular film is formed on only a specific part of the substrate, the molecular film does not have to be formed in a sea-island structure; as another example, it may be formed in a striped pattern.

[0029] Molecular films are typically formed at the interface between the first surface of the substrate and the liquid phase. However, the disclosure is not limited to this configuration, and they may also be formed at the interface between the first surface of the substrate and the gas or solid phase.

[0030] Examples of molecular films include: lipid bilayers formed by hydrophilic-hydrophobic interactions; and metal-organic frameworks (MOFs) formed by the coordination of polyconformed organic molecules to polyconformed metal atoms.

[0031] Any molecule capable of self-assembling through non-covalent intermolecular interactions can be used as the membrane molecule. Examples of interactions acting between molecules for self-assembly include hydrophilic-hydrophobic interactions, electrostatic interactions, polyconformational coordination bonds, and polyconformational hydrogen bonds.

[0032] Examples of membrane molecules include: phospholipid molecules; combinations of polyconformate metal atoms and polyconformate organic molecules; nucleic acids such as RNA and DNA; and proteins that exhibit droplet-phase separation (LLPS). Among these, phospholipid molecules are preferred because they can form lipid bilayers and, due to their high biocompatibility, are suitable for capturing biomolecules.

[0033] Examples of phospholipid molecules that make up lipid bilayers include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (POPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOP Examples of phospholipid molecules include, but are not limited to, DOPC, 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt (POPS). In particular, the phospholipid molecule is preferably at least one selected from the group consisting of DOPC and DOPS. The phospholipid molecule may be one type or a combination of two or more types.

[0034] In lipid bilayers, the distance from each layer to the NV center differs by approximately 1 nm to 10 nm between the layer facing away from the first surface of the substrate (surface layer) and the layer facing the first surface of the substrate (back layer), typically by about 5 nm. This difference in distance results in a significant change in the signal intensity of quantum measurable elements such as spin present in or introduced by phospholipid molecules due to the NV center. Therefore, based on the signal intensity, it is possible to analyze whether the detected phospholipid molecule belongs to the surface layer or the back layer of the lipid bilayer. Consequently, it becomes possible to detect the movement phenomenon of phospholipid molecules between the surface layer and the back layer constituting the lipid bilayer, i.e., flip-flop. From the viewpoint of producing a more significant change in signal intensity, the number of carbon atoms in the straight-chain portion of the hydrocarbon chain of the phospholipid molecule is preferably 30 or more.

[0035] An example of a combination of metal atoms and organic molecules constituting a metal-organic structure is the combination of Zn(NO3)2 and terephthalic acid (H2(bdc)). By employing this combination, it is possible to form MOF-5 as a molecular film. As a technology related to MOFs, represented by MOF-5, a technology has recently been developed that applies MOF particles modified with biomolecules such as sugars or peptides to drug delivery. In one embodiment of this disclosure, employing MOF as a molecular film is useful for analyzing the dynamics of such biomolecularly modified MOFs.

[0036] The membrane molecules may be transported within the molecular membrane by biomolecules. By detecting the transport of membrane molecules by biomolecules using an NV center, it becomes possible to analyze the transport activity of biomolecules. In particular, it is preferable for the membrane molecules to be transported perpendicular to the first surface of the substrate in order to perform detection by the NV center with higher sensitivity.

[0037] The film molecule may have any type of label depending on the detection method using NV centers. Examples of labels include: spin labels detected by electron spin resonance (ESR) spectroscopy; fluorescent labels detected by fluorescence observation; and nuclear spin labels detected by nuclear magnetic resonance (NMR) spectroscopy. Among these, it is preferable for the film molecule to have a spin label from the viewpoint of having a small label structure and reducing the influence of the label on the dynamics of the film molecule. The spin label may be a known one, and an example of such a label is Gd 3+ Examples include the Doxyl group contained in 5-Doxyl-stearic acid or 16-Doxyl-stearic acid, etc.

[0038] (Biomolecules) The observed sample may further contain biomolecules trapped in the molecular film. In one embodiment of this disclosure, the biomolecules may be the subject of dynamic analysis. Alternatively, in one embodiment of this disclosure, the biomolecules may be trapped in the molecular film that is the subject of dynamic analysis and may interact with the molecular film in various forms, such as chemical reactions or molecular transport.

[0039] In this disclosure, "a biomolecule is trapped in a molecular film" means that the biomolecule exhibits a higher affinity for the molecular film than for the liquid, gaseous, or solid phase on the opposite side of the molecular film from the substrate, and that the biomolecule is reversibly brought into close proximity with the molecular film. The manner of trapping is not particularly limited. Examples of trapping include the biomolecule penetrating the molecular film, the biomolecule being embedded in the molecular film, or the biomolecule adhering to the surface of the molecular film. Furthermore, trapping may be achieved by non-covalent interactions between the biomolecule and the molecular film, such as hydrophilic-hydrophobic interactions, van der Waals forces, and electrostatic interactions.

[0040] In this disclosure, biomolecules refer to molecules that are naturally produced or metabolized in living organisms, or artificial mimics thereof. Examples of biomolecules include membrane proteins and droplet-forming proteins such as FUS, LAF-1, and TDP-43. Examples of membrane proteins include flippases such as ATP11C and P4-ATPase; and scramblases such as TMEM16 and XKR8. Examples of artificial mimics include amino acid sequence modifications of the above-mentioned membrane proteins, and modifications with detection labels. A biomolecule may be a single type or a combination of two or more types.

[0041] From the perspective of analyzing the dynamics of biomolecules in a state as close to nature as possible, when the biomolecule is a membrane protein, it is preferable that the membrane molecule is a phospholipid molecule. This results in a lipid bilayer composed of phospholipid molecules, with membrane proteins embedded within it, which suitably mimics a natural cell membrane.

[0042] The analysis of lipid molecule flip-flops by flippases, and the analysis of lipid molecule transport mechanisms by other membrane proteins, are deeply related to biological processes such as cell death, including apoptosis, and blood coagulation. Elucidating these mechanisms is expected to contribute to the development of novel therapeutic drugs. Biological phenomena involving membrane protein dynamics, such as lipid scrambling, include various biological phenomena such as cell death, blood coagulation, viral infection, synaptic pruning, fertilization, and myoblast fusion. In this disclosure, employing combinations of membrane proteins and phospholipid molecules is useful for elucidating these various biological phenomena.

[0043] Biomolecules may be transported within molecular membranes or form aggregates with other biomolecules within molecular membranes. By detecting the transport or aggregation of biomolecules using an NV center, it becomes possible to analyze the activity of biomolecule transport and aggregation. An example of a biomolecule that forms aggregates is the Atg protein. Atg proteins are known to assemble near vacuolar membranes in vivo to form preautophagosome structures (PAS). In one embodiment of this disclosure, employing Atg proteins is useful for analyzing the dynamics of PAS. In addition to Atg proteins, proteins that generate liquid-liquid phase separation (LLPS) in cells may also be employed, which is biologically useful for analyzing the dynamics of interactions between LLPS and various molecular membranes in cells.

[0044] Biomolecules may have any type of label depending on the detection method using NV centers. Examples of labels include: spin labels detected by electron spin resonance (ESR) spectroscopy; nuclear spin labels detected by nuclear magnetic resonance (NMR) spectroscopy; and fluorescent labels detected by fluorescence observation. In particular, biomolecules are preferred to have spin labels because the label structure is small and the influence of the label on the dynamics of the biomolecule is reduced. Spin labels may be known, and an example is Gd 3+Examples include the Doxyl group contained in 5-Doxyl-stearic acid or 16-Doxyl-stearic acid, etc.

[0045] (Auxiliary agent) When the observed sample contains biomolecules, the sample may further contain auxiliary agents within the molecular film to promote the capture of biomolecules by the molecular film. These auxiliary agents function to regulate the interaction between biomolecules and the molecular film, and are typically present at the interface between biomolecules and membrane molecules in the observed sample. The auxiliary agents should be appropriately selected depending on the combination of biomolecules and the molecular film.

[0046] For example, when the membrane molecule is a phospholipid molecule and the biomolecule is a membrane protein, it is preferable that the observation sample further contains a surfactant as an auxiliary agent in the molecular membrane. The surfactant interposes between the hydrophilic group on the surface of the membrane protein and the hydrophobic hydrocarbon group of the phospholipid molecule in the structure of the lipid bilayer and the membrane protein that penetrates it, stabilizing the penetration state.

[0047] Examples of surfactants include, but are not limited to, sodium dodecyl sulfate (SDS), octyl β-glucoside (OG), n-dodecyl-β-D-maltoside (DDM), 3-((3-colamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), n-dodecylphosphocholine (DPC), 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (LMPG), lauryl maltose neopentyl glycol (LMNG), and decyl maltose neopentyl glycol (DMNG).

[0048] Other examples of auxiliary agents include terephthalic acid solvent (Zn(NO3)2-4H2O), which is used in the formation of MOF-5.

[0049] (Application) An observation sample according to one aspect of this disclosure can be subjected to known analytical methods that use an NV center as a quantum sensor. Examples of analytical methods include: nanoscale electron spin resonance (ESR) spectroscopy; nanoscale nuclear magnetic resonance (NMR); detection using relaxation times or coherence times such as T1 and T2; and the like.

[0050] [Method for preparing observation samples] The observation sample can be prepared by incubating a buffer solution containing membrane molecules and, if necessary, biomolecules and auxiliary agents, in contact with the first surface of a substrate. During incubation, the membrane molecules self-assemble on the first surface of the substrate, forming a molecular film, and the biomolecules and auxiliary agents are trapped in the molecular film. After incubation, it is preferable to remove any excess membrane molecules that do not constitute the molecular film by gently exchanging the solution with a buffer solution that does not contain membrane molecules, etc.

[0051] When using auxiliary agents, incubating the biomolecules and auxiliary agents beforehand, and then further incubating them with membrane molecules, will further promote the capture of biomolecules by the molecular membrane.

[0052] When forming a molecular film in an island-like structure, it is preferable to mask the portion of the first surface that forms the sea-like region and perform incubation. The mask used should preferably have surface properties that prevent film molecules from self-assembling on its surface. For example, when using phospholipid molecules as the film molecules, it is preferable to select a mask with a hydrophobic surface so that a lipid bilayer does not form on the mask surface.

[0053] 〔kit〕 One aspect of this disclosure relates to a kit for preparing an observation sample according to one aspect of this disclosure. The kit includes a substrate and instructions. The substrate included in the kit is identical to the substrate described above for the observation sample, and therefore a detailed description thereof will not be repeated.

[0054] The instructions describe to the user the procedure for forming a molecular film containing trapped biomolecules along the surface of the substrate. An example of the procedure described is the method for preparing the observation sample described above.

[0055] The instruction manual may be a paper document with instructions printed on it, or it may be an electronic document that stores the instructions for the user. Alternatively, instead of directly explaining the operation, the instruction manual may indirectly explain the operation by providing a link to access instructions (for example, a URL to an online document explaining the operation).

[0056] The kit may further include any of the following: membrane molecules, biomolecules, and auxiliary agents. The kit may also further include any of the following: a mask for forming molecular films in island-like structures, and a buffer solution, which are used in the method for preparing the observation sample.

[0057] To prepare observation samples using the kit, users may use the membrane molecules, biomolecules, and auxiliaries included in the kit, or prepare them themselves as needed.

[0058] [Embodiment 1] One embodiment of the present disclosure will be described with reference to Figure 1. Figure 1 is a schematic diagram showing the configuration of an observation sample 100 according to Embodiment 1 of the present disclosure. The observation sample 100 according to Embodiment 1 is used for the dynamics analysis of biomolecules.

[0059] As shown in Figure 1, the observation sample 100 comprises a substrate 11 having a diamond crystal layer 111 on which NV centers 111c are arranged, a molecular film 12 formed along the surface of the substrate 11 and composed of self-assembled molecules, and biomolecules 13 trapped in the molecular film 12.

[0060] In this embodiment, the substrate 11 has a diamond plate without NV centers as a second layer 112, and a diamond crystal layer formed on the second layer 112 as a first layer 111. The NV centers 111c are located at a depth T from the upper surface (first surface) of the first layer 111. In this embodiment, the depth T is 10 nm.

[0061] In this embodiment, the molecular membrane 12 is a lipid bilayer composed of phospholipid molecules. In the lipid bilayer, the phospholipids self-assemble so that hydrophilic phosphate groups are exposed on both sides. The biomolecule 13 is the flippase ATP11C. The biomolecule 13 penetrates the molecular membrane 12. From the viewpoint of suitably forming a complex between the flippase ATP11C and the lipid bilayer, in this embodiment, the phospholipid molecule is at least one selected from the group consisting of DOPC and DOPS.

[0062] In this embodiment, the biomolecule 13, which is a flippase, transports phospholipid molecules between the surface and back layers of the lipid bilayer, i.e., performs a flip-flop. Here, the detection region 111r by the NV center 111c covers the entire phospholipid molecule belonging to the back layer of the molecular film 12, as well as only the hydrocarbon chain of the phospholipid molecule belonging to the surface layer. Therefore, the spin label Gd coordinates to the hydrophilic phosphate group of the phospholipid molecule. 3+ This signal is detected strongly when phospholipid molecules are located on the underside of the molecular film. Therefore, by detecting spin labels, it becomes possible to analyze the flip-flop operation mediated by biomolecules 13.

[0063] Observation sample 100 further contains a surfactant 14 within the molecular film 12. The surfactant 14 has hydrophilic and hydrophobic groups, and is located at the interface between the biomolecule 13 and the molecular film 12 such that the hydrophilic group is directed toward the biomolecule 13 and the hydrophobic group is directed toward the hydrocarbon chain of the phospholipid molecule. The surfactant 14, positioned in this manner, stabilizes the state in which the biomolecule 13 penetrates the molecular film 12. From the viewpoint of further stabilizing the complex between the flippase ATP11C and the lipid bilayer, the surfactant in this embodiment is LMNG.

[0064] The observation sample 100 has multiple island-like regions IR where the molecular film 12 is formed in an island-like manner, and sea-like regions SR surrounding the island-like regions IR where the molecular film 12 is not formed. The island-like regions IR are circular in shape with a diameter DI when viewed from a direction perpendicular to the substrate 11. In this embodiment, the diameter DI is 30 μm. The width DS of the sea-like regions SR is 20 μm.

[0065] In this embodiment, the amount of biomolecules 13 contained in the entire observation sample 100 is controlled to be small, and the number of biomolecules 13 captured in a single island-like IR region is either 1 or 0. By controlling the amount of biomolecules 13 in this way, it becomes possible to analyze the flip-flop activity of a single biomolecule 13.

[0066] The preparation method S100 for the observation sample 100 according to this embodiment will be described with reference to Figure 2. Figure 2 is a schematic diagram showing the preparation method S100 for the observation sample 100 shown in Figure 1.

[0067] In the preparation method S100, first the substrate 11 is prepared (step S11).

[0068] Next, a mask 200 is stacked on the first layer 111 (step S12). The mask 200 has a plurality of through holes 201. The through holes 201 are circular with a diameter DI and are spaced apart with a width DS.

[0069] A buffer solution containing membrane molecules 12a, biomolecules 13, and surfactant 14 is poured into the laminate (step S13). When the laminate is incubated, a molecular film 12 is formed in the diamond crystal layer 111 at the bottom of the through-hole 201 by the self-assembly of membrane molecules 12a. Biomolecules 13 are trapped in the molecular film 12 together with the surfactant 14 (step S14).

[0070] When a buffer solution that does not contain membrane molecules 12a etc. is poured into the laminate, excess membrane molecules 12a etc. that were suspended in the liquid phase are removed (step S15). Finally, when the mask 200 is removed, an observation sample 100 having island-like IR regions and sea-like SR regions is obtained (step S16).

[0071] [Embodiment 2] Embodiment 2 of this disclosure will be described below. For the sake of clarity, components having the same function as those described in the above embodiment will be denoted by the same reference numerals, and their descriptions will not be repeated.

[0072] The observation sample 600 according to this embodiment will be described with reference to Figure 3. Figure 3 is a schematic diagram showing the configuration of the observation sample 600 according to Embodiment 2 of this disclosure. The observation sample 600 according to this disclosure is used for the dynamic analysis of molecular films.

[0073] As shown in Figure 3, the observation sample 600 has a substrate 11 having a diamond crystal layer 111 on which NV centers 111c are arranged, and a molecular film 12 formed along the surface of the substrate 11 and composed of self-assembled molecules, and has at least one island-like region IR on the surface of the substrate 11 in which the molecular film 12 is formed in an island-like manner, and a sea-like region SR surrounding the at least one island-like region IR, on the surface of the substrate 11 where no molecular film 12 is formed.

[0074] [Additional Notes] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0075] 〔summary〕 As can be understood from the above description, this disclosure encompasses the following aspects:

[0076] Embodiment 1: An observation sample (100) for analyzing the dynamics of a biomolecule (13), characterized by comprising: a substrate (11) having a diamond crystal layer (111) on which NV centers (111c) are arranged; a molecular film (12) formed along the surface of the substrate and composed of self-assembled molecules (12a); and the biomolecule captured in the molecular film. According to this embodiment, a complex of a molecular film formed by self-assembly and a biomolecule captured in the molecular film can be subjected to dynamic analysis by an NV center acting as a quantum sensor. While the molecular film as a whole maintains a film-like structure, individual film molecules exhibit fluidity, making it possible to analyze the dynamics of biomolecules within a minute volume using the NV center. Thus, an observation sample is provided for analyzing the minute dynamics of biomolecules in a near-natural state.

[0077] Embodiment 2: An observation sample according to Embodiment 1, characterized in that the observation sample has at least one island-like region (IR) on the surface of the substrate in which the molecular film is formed in an island-like manner, and a sea-like region (SR) surrounding the at least one island-like region on the surface of the substrate in which the molecular film is not formed. According to this embodiment, the range in which film molecules can move in a direction parallel to the surface of the substrate is limited to the island-like region. This limitation makes it possible to evaluate the distance and direction of movement of film molecules more quantitatively compared to the case in which the molecular film is formed over the entire first surface of the substrate.

[0078] Embodiment 3: An observation sample according to Embodiment 1 or 2, characterized in that the biomolecule or the molecules constituting the molecular membrane are transported within the molecular membrane. According to this embodiment, the transport activity of biomolecules or membrane molecules can be analyzed by detecting the transport of biomolecules or membrane molecules with an NV center.

[0079] Embodiment 4: An observation sample according to any one of Embodiments 1 to 3, characterized in that the molecules constituting the molecular membrane are phospholipid molecules and the biomolecules are membrane proteins. According to this embodiment, an observation sample is provided which has a lipid bilayer composed of phospholipid molecules, with membrane proteins embedded in it, and which suitably mimics a natural cell membrane.

[0080] Embodiment 5: An observation sample according to any one of Embodiments 1 to 4, characterized in that the observation sample further contains a surfactant in the molecular film. According to this embodiment, the surfactant interposes between the hydrophilic group on the surface of the membrane protein and the hydrophobic hydrocarbon group of the phospholipid molecule in the structure of the lipid bilayer and the membrane protein captured therein, thereby stabilizing the capture state.

[0081] Embodiment 6: An observation sample according to Embodiment 4 or 5, characterized in that the phospholipid molecule has a hydrocarbon chain with 30 or more carbon atoms in the linear portion. According to this embodiment, it becomes easier to detect the movement of phospholipid molecules between the surface and back layers constituting the lipid bilayer, i.e., flip-flop.

[0082] Embodiment 7: An observation sample according to any one of Embodiments 1 to 6, characterized in that at least one of the biomolecules and molecules constituting the molecular film has a spin label. According to this embodiment, since spin labels have a smaller structure compared to fluorescent labels detected by fluorescence observation, the influence of the label on the dynamics of biomolecules or membrane molecules is reduced, and an observation sample is provided for analyzing the minute dynamics of biomolecules or membrane molecules in a state closer to nature.

[0083] Embodiment 8: A kit for preparing an observation sample according to any one of Embodiments 1 to 7, comprising a substrate and instructions, wherein the instructions describe to the user the operation for forming the molecular film on which the biomolecules are captured along the surface of the substrate. According to this embodiment, a kit is provided for analyzing the minute dynamics of biomolecules in a near-natural state.

[0084] Embodiment 9: An observation sample (600) for dynamic analysis of a molecular film (12), comprising: a substrate (11) having a diamond crystal layer (111) on which NV centers (111c) are arranged; and the molecular film (12) formed along the surface of the substrate and composed of self-assembled molecules, wherein the observation sample comprises at least one island-like region (IR) on the surface of the substrate where the molecular film is formed in an island-like manner, and a sea-like region (SR) surrounding the at least one island-like region, on the surface of the substrate where the molecular film is not formed. According to this embodiment, the range in which film molecules can move in a direction parallel to the surface of the substrate is limited to the island-like region. This limitation makes it possible to evaluate the distance and direction of movement of film molecules more quantitatively compared to the case in which the molecular film is formed over the entire first surface of the substrate. While the molecular film as a whole maintains a film-like state, individual film molecules exhibit fluidity, and the dynamics of film molecules within a minute volume can be analyzed by the NV centers, thus providing an observation sample for analyzing the minute dynamics of a self-assembled molecular film in a near-natural state. [Examples]

[0085] An embodiment of this disclosure is described below. In the following embodiment, a complex containing a lipid bilayer and ATP11C, a membrane protein flippase that penetrates the bilayer, was formed on a diamond crystal layer having NV centers. The formation of the complex was verified using fluorescence observation such as photobleaching-resulting fluorescence recovery (FRAP), and spin labels present in the lipid bilayer were detected by quantum measurement. This demonstrated that the dynamics of ATP11C can be analyzed using nanoscale quantum measurement.

[0086] (Preparation of the diamond crystal layer) NV centers were formed in the region of 10 nm or less on the surface of a diamond plate by ion implantation and annealing. Furthermore, by acid treatment of this diamond plate, oxygen terminals were formed on the surface of the diamond crystal layer, creating a hydrophilic surface.

[0087] (Formation of lipid bilayer-ATP11C complex and verification by fluorescence observation) ATP11C was prepared by adjusting the concentration of a pre-prepared solution with buffer to obtain an ATP11C solution. 1: The following lipids and spin labels were mixed in a light-shielding bottle to prepare the solution. DOPC 1200 μL (12 mg) DOPS 300 μL (3 mg) ·0.15mg Rhodamine Red 150μL(stock 1mg / ml) ·0.15mg Gd 150μL(stock 1mg / ml) The mixture was dispensed into light-shielding bottles in 112 μL (Gd 0.01 mg) portions, and the bottles were vacuumed to allow for long-term storage. 2: Dissolve the DOPC in 500 μL of the buffer described below in a light-shielding bottle (DOPC 1 mg / 500 μL). 3. 200 μL (0.4 mg) was taken from the light-shielding bottle, and 6 μL (0.02 mg) of GFP-ATP11C (3.3 mg / ml) was added. In this study, the ratio of lipids to membrane proteins (GFP-ATP11C) was 20:1. 4. The solution was ice-sonicated for 5 minutes. 5. Place the PDMS pattern and coverslip, immersed in 100% ethanol, onto a diamond surface free of dust and other debris, and allow to dry at room temperature for 5 minutes. 6. Cut the cover slip in the area where the pattern was placed to approximately 5mm x 5mm using a diamond pen and place it in a single-well dish. 7:500 μL of 30% ethanol was placed in a single well to remove air bubbles that had formed in the pattern. 8. The ethanol was changed several times to completely remove the bubbles. 9. After washing the PDMS 10 times with water, 200 μL of lipid + GFP-ATP11C prepared in step 4 above was added and incubated for 30 minutes. 10: Washed 10 times with water (at this point, the coverslip floated and the solution was exposed to air, so half the water was changed each time to avoid this). 11: Observations were made using a microscope and an atomic force microscope (AFM).

[0088] Buffer composition 5M NaCl 1mL 2M MgCl 20.1mL 0.5M MES / pH6.5 4mL • Add pure water and make up to 100 mL.

[0089] The formation of a lipid bilayer-ATP11C complex on the diamond crystal layer was verified by fluorescence observation. Specifically, a 40x objective lens (LCACHN40XIPC, Olympus) was used, and the observation sample was excited with a dichroic mirror laser (wavelength 532 nm for rhodamine, wavelength 488 nm for GFP), and fluorescence was detected with a camera. The results are shown in Figures 4(a) and (b). As shown in Figures 4(a) and (b), island-like rhodamine-phospholipid molecule films were formed, and the presence of ATP11C within these films was observed.

[0090] Next, the sample was observed using FRAP. Specifically, the excitation spot was narrowed to a portion of one island-shaped region, and laser excitation with a wavelength of 532 nm was applied at an intensity of approximately 10 5 mW·cm -2 The procedure was performed for 5 minutes to induce photobleaching of rhodamine, and the recovery of fluorescence was observed over time. The results are shown in Figure 4(c). As shown in Figure 4(c), a gradual recovery of fluorescence was observed from the periphery of the faded spot. This indicates that the lipid bilayer is formed in a manner in which the phospholipid molecules are fluid. Therefore, the formation of the lipid bilayer-ATP11C complex was demonstrated.

[0091] The excitation spot was further narrowed compared to the observation shown in Figure 4, and FRAP was performed again. In this experiment, instead of ATP-11C, a complex obtained by binding CDC50A to ATP-11C was used. The results are shown in Figure 6. As shown in Figure 6(a), it was observed that fluorescence gradually recovered from the periphery of the faded spot. This can also be seen in Figure 6(b). Furthermore, as shown in Figure 6(c), it was observed that a membrane of rhodamine-phospholipid molecules was formed in an island-like manner, and that GFP-ATP11C was evenly distributed within this membrane.

[0092] The formation of a lipid bilayer-ATP11C complex on a diamond crystal layer was verified by AFM observation. The results are shown in Figure 7. As shown in Figure 7(a), ATP11C was observed by AFM. In Figure 7(b), molecules thought to be monomer-derived were observed with a height of approximately 6-7 nm. Furthermore, a structure with a height of approximately 15 nm, about twice as tall, was also confirmed, suggesting that this structure may be a dimer. In Figure 7(c), black arrows indicate higher locations, and white arrows indicate locations that are tall but not as tall as those indicated by black arrows. Here, it is known that ATP11C protrudes at different distances to the sides of the cell membrane, protruding 2 nm towards the extracellular space and 7 nm towards the cytoplasm. Therefore, it is thought that the height also changes depending on the orientation in which ATP11C is compounded with the lipid bilayer. Specifically, the area indicated by the black arrow is thought to be where the cytoplasmic side of ATP11C is located in front of the lipid bilayer, and the area indicated by the white arrow is thought to be where the extracellular space side of ATP11C (i.e., the side to which the CDC50A domain binds to ATP11C) is located in front of the lipid bilayer. As described above, Figure 7(c) shows that it is possible to distinguish the orientation of ATP11C using AFM.

[0093] (Verification of the possibility of observing the dynamics of ATP11C using nanoscale quantum measurement ESR) Figure 5 shows the results of quantum measurement. Figure 5(a) shows the results of observation using conventional light. It is possible to identify the region where the lipid bilayer exists. Furthermore, Figure 5(b) shows an image of the effect of spins contained in lipid molecules obtained by imaging using quantum measurement. More specifically, as shown in Figure 5(c), a decrease in relaxation time (here, time T1) was confirmed in the region where the lipid bilayer exists due to the effect of spins. This phenomenon can be normalized as a quantum measurement within the measurement sequence and can be replaced with the quantitative number of spins present. From these results, it was confirmed that the effect of spins present in the lipid bilayer was confirmed, and that the change in the position of these spins in the surface and back layers of the lipid bilayer causes the change in relaxation time observed in Figure 5(c). These results verified that it is possible to identify flip-flops.

[0094] The results described above demonstrate that the observation samples in this embodiment allow for the analysis of minute dynamics of self-assembled molecular membranes such as lipid bilayers, and biomolecules such as membrane proteins, while maintaining fluidity close to that of natural materials. [Industrial applicability]

[0095] This disclosure can be used to analyze the dynamics of various molecules present in living organisms. [Explanation of Symbols]

[0096] 11 circuit boards 12 Molecular membrane 13 Biomolecules 14. Surfactants 100 observation samples 111 First layer (diamond crystal layer) 111c NV Center 111r detection area 112 Second layer (diamond plate) 200 masks 201 Through hole IR island area SR Oceanic region

Claims

1. Observation samples for analyzing the dynamics of biomolecules, A substrate having a diamond crystal layer on which NV centers are arranged, A molecular film composed of self-assembled molecules formed along the surface of the substrate, The biomolecule captured in the molecular film, An observation sample characterized by the following features.

2. The aforementioned observation sample is At least one island-like region on the surface of the substrate in which the molecular film is formed in an island-like manner, The system comprises a sea-like region surrounding the at least one island-like region, on the surface of the substrate, where the molecular film is not formed. The observation sample according to feature 1.

3. Within the molecular film, the biomolecules, or the molecules constituting the molecular film, are transported. The observation sample according to feature 1 or 2.

4. The molecules constituting the molecular film are phospholipid molecules, The aforementioned biomolecule is a membrane protein. The observation sample according to feature 1 or 2.

5. The aforementioned observation sample further contains a surfactant in the molecular film. The observation sample according to feature 4.

6. The phospholipid molecule has a hydrocarbon chain in which the number of carbon atoms in the linear portion is 30 or more. The observation sample according to feature 4.

7. At least one of the biomolecules and the molecules constituting the molecular film has a spin label. The observation sample according to feature 1 or 2.

8. A kit for preparing an observation sample according to claim 1 or 2, The circuit board and the instruction manual are included. The above-mentioned instruction manual explains to the user the procedure for forming the molecular film in which the biomolecules are trapped along the surface of the substrate. A kit characterized by the following features.

9. A sample for observation of molecular film dynamics analysis, A substrate having a diamond crystal layer on which NV centers are arranged, The present invention comprises a molecular film composed of self-assembled molecules formed along the surface of the substrate, At least one island-like region on the surface of the substrate in which the molecular film is formed in an island-like manner, The system comprises a sea-like region surrounding the at least one island-like region, on the surface of the substrate, where the molecular film is not formed. An observation sample characterized by the following features.

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