Probe with mesoporous metal formed thereon

Abstract

The purpose of the present invention is to newly provide a probe with mesoporous metal formed thereon. A probe with mesoporous metal formed thereon.

Description

probe formed from mesoporous metal

[0001] The present invention relates to a probe formed from mesoporous metal.

[0002] Identifying the distribution of molecules (such as proteins) on the surface and inside cells is crucial for elucidating cell function.

[0003] Atomic force microscopy (AFM) enables the measurement of sample shape at the nanometer level using a sharp probe tip. Furthermore, by forming a precious metal that exhibits plasmon resonance, such as gold, on the surface of the probe, molecular identification of the sample by Raman spectroscopy becomes possible. This probe is a technique that contributes to identifying the distribution of molecules on the surface and inside cells.

[0004] Conventionally, in Raman spectroscopy measurements using AFM, when only a metal film is formed on the probe, the intensity of Raman scattering is weak, making it difficult to detect trace amounts of molecules. Furthermore, when forming a metal film on the probe using deposition methods such as sputtering, the probe diameter increases in proportion to the film thickness, leading to problems such as a decrease in the resolution of shape measurements.

[0005] The present invention aims to provide a new probe made of mesoporous metal.

[0006] This invention develops a molecular measurement technique using an innovative probe (preferably an AFM probe) that achieves both high resolution and high sensitivity Raman spectroscopy measurements by forming a porous nanometal film (mesoporous metal film) on the probe, compared to conventional probes (such as AFM probes).

[0007] This invention enables the identification of molecular distribution on the surface and inside cells.

[0008] The present invention encompasses probes formed from the following mesoporous metal.

[0009] Item 1. A probe having a metal with a mesoporous structure on its surface, wherein (A) the back surface has a structure in which the substrate is exposed, or (B) the back surface has a structure in which the metal with the mesoporous structure is absent.

[0010] Item 2. The probe according to Item 1, wherein the surface is at least a portion of the surface of the tip of the probe.

[0011] Item 3. The probe described in Item 1, wherein the back surface has an exposed thermally oxidized SiO2 surface and forms an insulating film.

[0012] Item 4. The probe according to Item 1, wherein the metal is composed of at least one element selected from the group consisting of gold, silver, and copper.

[0013] Item 5. The probe according to Item 1, wherein the probe tip has the mesoporous structure on the surface of the tip, the mesoporous structure is a three-dimensional nanostructure having pores with a diameter of 2 nm to 50 nm, and the tip has a structure capable of puncturing a cell membrane.

[0014] Item 6. The probe according to Item 1, wherein the tip has the shape of a triangular pyramid, a square pyramid, or a cone, and the tip is structured to be able to puncture a cell membrane.

[0015] Item 7. The probe according to Item 1, wherein the tip diameter is 5 nm to 300 nm, and the tip has a structure that can puncture a cell membrane.

[0016] Item 8. The probe described in Item 1, wherein the probe is a probe used for Raman spectroscopy and / or a probe used for shape measurement by an atomic force microscope.

[0017] Item 9. A method for manufacturing a probe, comprising: electroplating the surface of the probe in an electrolyte containing a surfactant and metal ions by an electroplating method; reducing and depositing the metal on the surface of the probe to form a metal having a mesoporous structure on the surface of the probe.

[0018] The probe (preferably an AFM probe) of the present invention enables high-resolution and high-sensitivity Raman spectroscopy measurements in molecular measurement techniques by forming a porous nanometal film (mesoporous metal film) on the probe.

[0019] The probe of the present invention enables the identification of molecular distribution on the surface and inside cells.

[0020] The present invention provides a new probe made of mesoporous metal.

[0021] Figure 1 shows a method for fabricating a probe (AFM probe) having a mesoporous Au film. Figure 2 shows a probe with a mesoporous Au film fabricated and formed under the conventional solution concentration (Au: 5 mM) and electroplating time (30 min). Figure 3 shows a probe with a mesoporous Au film fabricated and formed under the conditions of an Au ion concentration of 5 mM and electroplating times of (a) 10 min, (b) 5 min, and (c) 0.5 min. Figure 4 shows a probe with a mesoporous Au film fabricated and formed under the conditions of an Au ion concentration of 250 μM (1 / 20 times the concentration of the conventional technology) and a plating time of 0.5 min (30 sec) (1 / 60 times the concentration of the conventional technology). Figure 5 shows the results of Raman spectroscopy measurements of a probe with a mesoporous Au film formed on it, after drying and fixing Nile blue onto the probe. Figure 6 shows the results of fabricating mesoporous Au films by changing the amount of organic solvent (tetrahydrofuran, hereafter THF) used and the molecular weight of the polymer, and the probe with the mesoporous Au film formed on it, as well as the change in the pore size of the probe. Figure 7 shows an SEM image of the back side of the probe with the mesoporous Au film formed on it. Figure 8 shows the results of Raman spectroscopy measurements (1 mM Nile blue) using a mesoporous Au nano probe (example) and a nonporous Au nano probe (comparative example). Figure 9 shows the field of ultra-precision nanofabrication and ultra-high sensitivity nanomeasurement opened up by mesoporous metal AFM.

[0022] The present invention will be described in detail below. The embodiments illustrating the present invention are intended to provide a better understanding of the spirit of the invention and do not limit the scope of the invention unless otherwise specified.

[0023] In this specification, "contains" and "include" are concepts that encompass all of the following: "comprise," "consist essentially of," and "consist of."

[0024] In this specification, when a numerical range is indicated as "A to B", it means "A or more and B or less".

[0025] In this specification, generally, the notations such as parts, % etc. are used to represent parts by mass, parts by weight, mass %, and weight % (wt%).

[0026] [1] Method for manufacturing a probe with a mesoporous metal formed thereon (FIG. 1) The method for manufacturing the probe of the present invention depends on an electroplating method. In an electrolytic solution containing a surfactant and metal ions, the surface of the probe is electrolytically plated, and the metal is reduced and deposited on the surface of the probe to form a metal having a mesoporous structure (pores of 2 nm to 50 nm) on the surface of the probe.

[0027] According to the method for manufacturing the probe of the present invention, by imparting a mesoporous structure to the surface of the probe, it is possible to enhance the Raman intensity by about 1.4 to 1.5 times.

[0028] (FIG. 1) FIG. 1 shows the manufacturing procedure of the probe (probe, AFM probe) with the mesoporous metal of the present invention formed thereon.

[0029] Step (1) First, the surface of a silicon (Si) probe (AFM probe etc.) (substrate) is heated to 950 °C using, for example, a muffler furnace, and a SiO2 film is formed by thermal oxidation. The silicon (Si) probe (AFM probe etc.) is the substrate portion of the probe of the present invention.

[0030] The probe of the present invention has a metal having a mesoporous structure on its surface, and (A) the back surface has a structure in which the substrate (silicon) is exposed, or (B) the back surface has a structure in which the metal having the mesoporous structure does not exist.

[0031] The back surface of the probe of the present invention exposes a thermally oxidized SiO2 surface (SiO2 film), which forms an insulating film.

[0032] Step (2) Next, on the surface of the probe (silicon substrate), for electrolytic plating, an adhesion layer and a conductive layer are formed by sputtering. The adhesion layer is preferably formed of Cr (chromium, 10 nm), Ti (titanium, 10 nm), etc. The conductive layer is preferably formed of Pt (platinum, 20 nm), Ag (silver, 20 nm), Au (gold, 20 nm), etc.

[0033] The surface of the probe of the present invention is at least a part of the surface of the tip of the probe.

[0034] Step (3) Next, the surface of the probe is electrolytically plated in an electrolytic solution containing a surfactant (polymer micelle (PS-b-PEO)) and metal ions. By this, metal (such as Au) ions are drawn into the micelles while reducing and depositing metal (such as Au) on the surface of the probe.

[0035] (Electrolytic plating method) The electrolytic plating method uses a micellized surfactant (soft template) and synthesizes mesoporous metal (metal film) by an electrochemical deposition method.

[0036] (Surfactant, formation of micelle solution) The surfactant is a component that forms a micelle (soft template) solution, and an amphiphilic block copolymer is used. The amphiphilic block copolymer refers to a block copolymer containing a hydrophobic part and a hydrophilic part.

[0037] The amphiphilic block copolymer may be a block copolymer of a hydrophobic polymer and a hydrophilic polymer, or a block copolymer of two different polymers containing a hydrophobic part and a hydrophilic part. From the viewpoint of micelle-forming ability, etc., preferably, a block copolymer of a hydrophobic polymer and a hydrophilic polymer is used.

[0038] The amphiphilic block copolymer may be a diblock copolymer composed of two polymers, or a triblock copolymer or more composed of three or more blocks. From the viewpoint of availability, etc., preferably, a diblock copolymer or a triblock copolymer is used.

[0039] Next, we will explain the case where the amphiphilic block copolymer is a block copolymer of a hydrophobic polymer and a hydrophilic polymer.

[0040] The hydrophobic polymer is not particularly limited. Examples of hydrophobic polymers include phenolic hydroxyl group-containing polymers such as polystyrene (also written as "PS"). Polystyrene is preferred as the hydrophobic polymer because it forms micelles that are more rigid and have superior function as a structure-controlling agent.

[0041] The hydrophilic polymer is not particularly limited. Examples of hydrophilic polymers include poly(oxyalkylenes) such as poly(oxyethylene) (also written as "PEO"); polysiloxanes such as dimethylpolysiloxane; carboxyl group-containing polymers such as polyacrylic acid and polymethacrylic acid; and poly(N-methyl-2-vinylpyridinium) iodide salt. Poly(oxyethylene) is preferred as the hydrophilic polymer because it tends to create a large difference in hydrophilicity between it and hydrophobic polymers, particularly polystyrene.

[0042] From the viewpoint of micelle-forming ability and other factors, a phenolic hydroxyl group-containing polymer-poly(oxyalkylene) block copolymer is preferably used for the block copolymer of a hydrophobic polymer and a hydrophilic polymer.

[0043] Specific examples of amphiphilic block copolymers include polystyrene-b-poly(oxyethylene), polystyrene-b-poly(2-vinylpyridine)-b-poly(oxyethylene), poly(2-vinylpyridine)-b-dimethylpolysiloxane, polystyrene-b-polyacrylic acid, and polystyrene-b-poly(N-methyl-2-vinylpyridinium)iodate. From the viewpoint of micelle-forming ability and other factors, polystyrene-b-poly(oxyethylene) is preferably used as the amphiphilic block copolymer. The "-b-" indicates that the polymers form a block copolymer with each other.

[0044] The molecular weight of the polymer constituting each block in the amphiphilic block copolymer is preferably 3,000 to 100,000, and more preferably 5,000 to 70,000, from the viewpoint of micelle-forming ability and the like.

[0045] When the amphiphilic block copolymer is a block copolymer of a hydrophobic polymer and a hydrophilic polymer, from the viewpoint of micelle-forming ability and the like, the molecular weight of the hydrophobic polymer is preferably 7,000 to 100,000, more preferably 10,000 to 70,000. The molecular weight of the hydrophilic polymer is preferably 3,000 to 50,000, more preferably 5,000 to 30,000.

[0046] First, a surfactant (such as a block copolymer of polystyrene-β-polyethylene oxide) is dissolved in tetrahydrofuran (THF) by sonication or heat treatment. Next, ethanol and deionized water are added to form a micelle solution (at room temperature to about 40°C). The pH of the micelle solution may be adjusted using hydrochloric acid to increase its conductivity.

[0047] (Metal materials, formation of mesoporous metals) The metal materials used to form mesoporous metals (metal films) are preferably platinum (Pt), palladium (Pd), rhodium (Rh), ruthenium (Ru), osmium (Os), iridium (Ir), gold (Au), silver (Ag), copper (Cu), rhenium (Re), mercury (Hg), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), zinc (Zn), gallium (Ga), niobium (Nb), molybdenum (Mo), cadmium (Cd), indium (In), tin (Sn), thallium (Tl), lead (Pb), bismuth (Bi), etc.

[0048] The metallic material is preferably a metallic material for fabricating probes used in Raman spectroscopy and / or probes used in shape measurement by atomic force microscopy. When the metallic material is used to fabricate probes for Raman spectroscopy, it is preferably a metal that exhibits plasmon resonance, and more preferably at least one element selected from the group consisting of gold, silver, and copper.

[0049] (Electrolyte for Electrodeposition) The electrodeposition electrolyte is adjusted to a deposition potential (e.g., -0.5V (vs. Ag / AgCl), for 30 seconds) and used as an electrolyte for typical electrodeposition of mesoporous films. A conventional three-electrode system is used for electrodeposition, using, for example, the following counter electrode, reference electrode, and working electrode (conductive substrate): Counter electrode: Pt wire Reference electrode: Ag / AgCl electrode (in 3M KCl) Working electrode: Probe with Pt (conductive layer) / Ti (adhesion layer) film (preferably an AFM probe)

[0050] Setting an appropriate deposition potential (reduction potential) is necessary to determine the deposition rate and structure of the mesoporous film.

[0051] It is also possible to set the current to achieve the appropriate potential (constant current electrolysis) and induce deposition.

[0052] (Metal ion concentration in electrodeposition electrolyte) In electrodeposition, current tends to concentrate on the protrusions, so the plating of probes requires optimization different from the general bath composition. The metal ion concentration in the electrodeposition electrolyte for manufacturing probes is preferably adjusted to a lower concentration than the conventional metal ion concentration (Au: 5 mM (mmol / L)) for the following reasons: (i) it is possible to sharpen the probe by changing the film thickness due to electroplating, (ii) it is possible to perform measurements in local areas (e.g., inside cells) using this probe, and (iii) it is possible to suppress thickening due to current concentration.

[0053] (Amount of organic solvent in electrodeposition electrolyte) The amount of organic solvent used in the electrodeposition electrolyte may preferably be adjusted to a smaller amount than the amount of organic solvent used in conventional technology (THF: 3 mL / electrodeposition electrolyte 8 mL), in order to (i) change the film thickness due to electroplating and sharpen the probe, and (ii) reduce the micelle particle size and enable local measurement (for example, inside cells, etc.) using this probe. The amount of organic solvent used in the electrodeposition electrolyte can be adjusted according to the application to change the pore size to a suitable size.

[0054] For example, by adjusting the solution to 3 mL of THF (tetrahydrofuran) per 8 mL of electrolytic electrolyte, or 2 mL of THF per 8 mL of electrolytic electrolyte, the tip of the probe can maintain a pointed shape. For example, by adjusting the solution to 1 mL of THF per 8 mL of electrolytic electrolyte, the size of the probe's hole can be reduced.

[0055] The amount of organic solvent used in the electrodeposition electrolyte should be adjusted according to the application, for example, to about 1 mL to 3 mL of organic solvent per 8 mL of electrodeposition electrolyte.

[0056] (Types of organic solvents in the electrodeposition electrolyte) The type of organic solvent in the electrodeposition electrolyte is preferably tetrahydrofuran (THF), N,N-dimethylformamide (DMF), 2-methyltetrahydrofuran (2-MeTHF), 1,3-dioxolane, tetrahydropyran (THP), 4,4-dimethyl-1,3-dioxane, etc., and more preferably THF, in order to (i) make it possible to sharpen the probe by changing the film thickness due to electroplating, and (ii) make it possible to reduce the micelle particle size and use this probe to perform measurements in local areas (for example, inside cells).

[0057] (Type of surfactant in electrolytic electrolyte) The type of surfactant (block copolymer) in the electrolytic electrolyte is preferably PS, in order to (i) make it possible to sharpen the probe by changing the film thickness due to electroplating, and (ii) reduce the micelle particle size, which enables localized measurements (e.g., inside cells) using this probe.5000 -b-PEO 2000、 PS 5000 -b-PEO 2200、 PS 10000 -b-PEO 4100 and PS 18000 -b-PEO 7500 and PS 63000 -b-PEO 26000 etc. are used, more preferably 、 PS 10000 -b-PEO 4100 and PS 18000 -b-PEO 7500 and PS 63000 -b-PEO 26000 etc. are used, still more preferably, PS 18000 -b-PEO 7500 and PS 63000 -b-PEO 26000 etc. are used.

[0058] (Electrolysis time (electroplating time) of electrodeposition) In electrodeposition, current tends to concentrate on convex portions. Therefore, for plating on the probe, deposition in a relatively short time is required. Under such conditions, the electrolysis time (electroplating time) of electrodeposition is adjusted to be shorter, preferably, compared to the electrolysis plating time (30 min (minutes)) of the prior art, from the viewpoints that (i) it enables the probe to be sharpened by changing the film thickness due to electroplating, and (ii) it enables measurement in a local area (for example, inside a cell, etc.) using this probe. The electrolysis time of electrodeposition is more preferably adjusted to about 0.5 min to 20 min, still more preferably adjusted to about 0.5 min to 10 min, and particularly preferably adjusted to about 0.5 min (1 / 60 times the time of the prior art) to 5 min.

[0059] The electrolysis time of electrodeposition is preferably adjusted to 10 seconds to 30 seconds because errors due to operation or errors due to batches are likely to occur with a short film formation time.

[0060] Electrodeposition allows for the sharpening of a probe while maintaining a porous structure by appropriately adjusting the electrolyte concentration and electroplating time. For example, by adjusting the metal ion concentration of the electrolyte to 250 μM and the electroplating time to 30 seconds (0.5 min), the film thickness due to electroplating can be changed. This probe can then be used for localized measurements (e.g., inside cells).

[0061] The probe manufactured by the method of manufacturing the probe of the present invention enables highly sensitive Raman peaks to be produced in Raman spectroscopy measurements due to the mesoporous structure, allowing for molecular identification. The probe manufactured by the method of manufacturing the probe of the present invention is expected to enable high-resolution shape measurement because the shape of a single nano-protrusion at the tip contributes to shape measurement. The probe manufactured by the method of manufacturing the probe of the present invention has a shape that enables both high resolution and high-sensitivity Raman spectroscopy measurements.

[0062] Step (4) Finally, the probe (AFM probe) is immersed in an organic solvent to remove micelles incorporated into the metal (Au, etc.) plating. This forms a mesoporous structure on the surface of the probe due to the particle size of the micelles. The surface of the probe has a metal that forms the mesoporous structure. The back surface (A) of the probe has a structure in which the substrate (silicon) is exposed. Alternatively, the back surface (B) of the probe has a structure in which the metal having the mesoporous structure is not present.

[0063] After electrodeposition, the working electrode is removed and immersed in THF at room temperature to remove the micelle template.

[0064] The surface of the probe is preferably at least a portion of the surface of the tip of the probe.

[0065] The metal that forms the mesoporous structure is preferably composed of at least one element selected from the group consisting of gold (Au), silver (Ag), and copper (Cu).

[0066] The mesoporous structure of the probe is preferably a three-dimensional nanostructure (porous structure) having pores with a diameter of 2 nm to 50 nm (mesoscale).

[0067] The probe tip is preferably shaped like a triangular pyramid, a square pyramid, or a cone.

[0068] Probes formed from mesoporous metal exhibit high Raman scattering intensity because the porous structure of the mesoporous metal, with its mesopores (2 nm to 50 nm), creates numerous electromagnetic field hotspots.

[0069] The probe (preferably an AFM probe) of the present invention enables high-resolution and high-sensitivity Raman spectroscopy in molecular measurement techniques by forming a porous nanometal film (mesoporous metal film) on the probe. The probe of the present invention enables identification of molecular distribution on the surface and inside of cells.

[0070] [2] Probe with mesoporous metal (Figures 2 to 8) The probe of the present invention has a metal having a mesoporous structure on its surface.

[0071] The probe of the present invention has a metal having a mesoporous structure on its surface, and (A) the back surface has a structure in which a substrate (silicon (Si) probe (AFM probe, etc.)) is exposed, or (B) the back surface has a structure in which the metal having the mesoporous structure is absent.

[0072] The probe of the present invention has a back structure that ensures the laser reflectance intensity for atomic force microscope (AFM) measurements.

[0073] The probe of the present invention has an exposed thermally oxidized SiO2 surface (silicon substrate) on its back surface, which acts as an insulating film. The absence of electroplating on the back surface of the probe maintains the laser reflectance intensity for AFM measurement.

[0074] The surface of the probe is preferably at least a portion of the surface of the tip of the probe.

[0075] The probe of the present invention preferably has a mesoporous structure on the surface of its tip. The mesoporous structure of the probe is preferably a three-dimensional nanostructure (porous structure) having pores with a diameter of 2 nm to 50 nm (mesoscale), and more preferably a diameter (pore size) of 2 nm to 32 nm. From the viewpoint of cell viability and puncture performance, the tip of the probe preferably has a structure that can puncture the cell membrane.

[0076] The metal of the probe is preferably composed of at least one element selected from the group consisting of gold (Au), silver (Ag), and copper (Cu).

[0077] The probe preferably has a tip shape that is a triangular pyramid, a square pyramid, or a cone. From the viewpoint of cell viability and puncture performance, the tip of the probe preferably has a structure that can puncture the cell membrane.

[0078] For cell puncture, the probe tip diameter (probe diameter) is preferably 5 nm to 300 nm, more preferably 5 nm to 200 nm, and even more preferably 5 nm to 160 nm, taking into consideration cell viability and puncture performance. The tip of the probe is preferably structured to be able to puncture the cell membrane, from the viewpoint of cell viability and puncture performance.

[0079] The probe is useful for Raman spectroscopy measurements. The probe is useful for shape measurements using an atomic force microscope (AFM).

[0080] The probe of the present invention enables highly sensitive Raman peaks to be produced in Raman spectroscopy measurements due to the mesoporous structure, allowing for molecular identification. The probe of the present invention is expected to enable high-resolution shape measurement, as the single nano-protrusion shape at the tip contributes to shape measurement. The probe of the present invention has a shape that enables both high resolution and high-sensitivity Raman spectroscopy measurements.

[0081] The probe of the present invention has a mesoporous structure on its surface, which makes it possible to increase the Raman intensity by approximately 1.4 to 1.5 times.

[0082] The probe (preferably an AFM probe) of the present invention enables high-resolution and high-sensitivity Raman spectroscopy in molecular measurement techniques by forming a porous nanometal film (mesoporous metal film) on the probe. The probe of the present invention enables identification of molecular distribution on the surface and inside of cells.

[0083] [3] Use of the probe with the mesoporous metal of the present invention The probe (preferably an AFM probe) of the present invention makes it possible to achieve both high resolution and high sensitivity Raman spectroscopy in molecular measurement technology by forming a porous nanometal film (mesoporous metal film) on the probe. The probe of the present invention makes it possible to identify the molecular distribution on the surface and inside of cells.

[0084] The present invention will be described in detail below based on examples.

[0085] The present invention is not limited by these embodiments.

[0086] [1] Manufacturing of a probe with mesoporous metal (Figure 1) Figure 1 shows the procedure for manufacturing a probe (AFM probe) with mesoporous metal according to the present invention.

[0087] First, an SiO2 film was formed on the surface of a Si probe (AFM probe) (substrate) by thermal oxidation by heating it to 950°C using a muffle furnace. Next, Ti (titanium, 10 nm, adhesion layer) and Pt (platinum, 20 nm, conductive layer) were deposited on the surface of the probe by sputtering. The Si (silicon) probe is the substrate portion of the probe. The back of the probe has a structure in which the substrate is exposed, and the thermally oxidized SiO2 surface is exposed (SiO2 film), forming an insulating film.

[0088] Next, the surface of the probe (working electrode) was electroplated in an electrolyte containing polymer micelles (PS-b-PEO) and Au (gold) ions (or Ag (silver) or Cu (copper) ions). This process reduced and deposited Au onto the probe surface, with Au ions drawing in the micelles.

[0089] Finally, the probe (AFM probe) was immersed in an organic solvent to remove micelles incorporated into the Au plating, thereby forming a mesoporous structure due to the particle size of the micelles. The probe has a metal surface with a mesoporous structure, while the back surface does not have a metal surface with a mesoporous structure.

[0090] Probes formed from mesoporous metal exhibit high Raman scattering intensity because the mesoporous metal, with its porous structure containing mesopores (2 nm to 50 nm), has many electromagnetic field hotspots.

[0091] [2] Evaluation of probes with mesoporous metal formed on them (Figure 2) Figure 2 shows the results of the conventional technology under the conditions of solution concentration (Au: 5 mM) and electroplating time (30 min) PS 18000 -b-PEO 7500 A mesoporous Au film was fabricated using the provided template, and the probe (AFM probe) on which this mesoporous Au film was formed is shown. A large number of particles approximately 2 μm to 3 μm in size were formed on the probe. The mesoporous structure was confirmed on the surface of the particles.

[0092] For cell puncture, a probe diameter of 200 nm or less is preferred, considering factors such as cell viability and puncture performance. Therefore, we investigated reducing the probe diameter by shortening the electroplating time when forming a mesoporous Au film on the probe. From the viewpoint of cell viability and puncture performance, the tip of the probe is preferably structured to be able to puncture the cell membrane.

[0093] (Figure 3) Figure 3 shows SEM images of the probe forming the mesoporous Au film when the Au ion concentration was kept constant at 5 mM and the electroplating time was reduced to 10 min, 5 min, and 0.5 min. When the electroplating time was reduced to 10 min, 5 min, and 0.5 min, the probe diameter of the probe forming the mesoporous Au film became smaller, at 4.3 μm, 3.1 μm, and 1.0 μm, respectively.

[0094] Next, we considered changing the film thickness due to plating by lowering the Au ion concentration.

[0095] (Figure 4) Figure 4 shows a probe (AFM probe) on which a mesoporous Au film was fabricated under the conditions of an Au ion concentration of 250 μM (1 / 20th the concentration of the conventional technology) and an electroplating time of 0.5 min (1 / 60th the time of the conventional technology).

[0096] A mesoporous Au film with a pore size of 32 nm was formed on the AFM probe. The tip diameter of the AFM probe was 160 nm, and the Au particles at the probe tip had a wall thickness of approximately 22 nm.

[0097] (Figure 5) Figure 5 shows the Raman measurement results of a probe with a soporous Au film formed on it, to which Nile Blue was dried and fixed. Nile Blue was dried and fixed on the fabricated probe (using the AFM probe in Figure 3(a)), and Raman spectroscopy was performed using a Raman microscope (laser wavelength: 532 nm). The result of the Raman spectroscopy was 590 cm⁻¹ -1 We confirmed that the characteristic Raman peak of Nile blue is highly sensitive due to the mesopore structure in the vicinity, making molecular identification possible.

[0098] This mesoporous Au-coated AFM probe is expected to enable high-resolution shape measurement because the single nano-protrusion at its tip is predicted to contribute to shape measurement. Thus, it has been found that the mesoporous Au-coated AFM probe has a shape that can achieve both high resolution and high-sensitivity Raman spectroscopy measurements.

[0099] Conventionally, it has been reported that reducing the amount of the organic solvent tetrahydrofuran (THF) used in electroplating the surface of a probe results in smaller micelle particle sizes.

[0100] (Figure 6) Figure 6 shows the results of preparing mesoporous Au films by changing the amount of organic solvent (THF) used and the molecular weight of the polymer, as well as the probes on which the mesoporous Au films were formed and the changes in the pore size of the probes. When the amount of THF used during electroplating of the probe surface was changed from a standard of 3 mL (Figure 4 (b-1)) to 2 mL and then to 1 mL, it was found that the particle size (probe diameter) formed on the probe also changed (Figure 6 (a), Figure 6 (b)).

[0101] When electroplating the surface of a probe, the molecular weight of the block copolymer is increased (PS 63000 -b-PEO 26000 ), the micelle particle size increased, and the pore size (probe diameter) formed also increased (Figure 6(c)).

[0102] When measuring the shape of cell samples or other specimens using an atomic force microscope (AFM), the shape of the sample is measured by shining a laser on the back of the probe and detecting the minute changes reflected back. When measuring shape using an AFM, uniformity of the back surface of the probe is important in order to ensure the laser reflection intensity. The probe has a metal surface with a mesoporous structure, and the back surface has a structure in which the substrate (silicon) is exposed, or a structure in which there is no metal with a mesoporous structure.

[0103] (Figure 7) Figure 7 shows an SEM image of the back side of a probe with a mesoporous Au film formed on it. The back side of the probe has an exposed thermally oxidized SiO2 surface (substrate) and acts as an insulating film, so electroplating is not performed and the laser reflectance intensity for AFM measurement is maintained.

[0104] (Example) Mesoporous Au probe (PS 18000 -b-PEO 7500 The Raman spectroscopy results are shown when a non-porous Au probe, electroplated with a solution (3 mL of THF) and (comparative example) a solution without polymer micelles, was approached on a dish with 1 mM Nile blue fixed in place. The experiment was repeated three times using a laser wavelength of 532 nm.

[0105] (Figure 8) Figure 8 shows the Raman spectroscopy results (1 mM Nile blue) using a mesoporous Au nanoprobe (example) and a nonporous Au nanoprobe (comparative example).

[0106] The comparative example, a non-porous Au nano probe, has a tip diameter of 160 nm and no pore size.

[0107] The mesoporous Au nanoprobe in the example has a tip of 160 nm and a pore diameter of 32 nm. The mesoporous Au nanoprobe in the example has a mesoporous structure (2 nm to 50 nm) at its tip, and the surface area of ​​the tip is larger than that of the nonporous Au nanoprobe in the comparative example.

[0108] Instrument: Atomic Force Microscope (AFM) Excitation Wavelength: 532nm Laser Output: 50mW Dimmer: Open (100%) Exposure Time: 10s Number of Accumulations: 10 Laser: Nile Blue A 5G Sigma-Aldrich (N0766-5G)

[0109] The results showed that imparting a mesoporous structure to the surface of the probe increased the Raman intensity by approximately 1.4 to 1.5 times.

[0110] [3] Industrial applicability of probes with mesoporous metals Conventionally, in order to achieve highly sensitive Raman spectroscopy measurements, it has been common to form convex nanoparticles near the probe as a processing method.

[0111] This invention is an unprecedented technology that reverses conventional processing methods by forming a concave mesoporous structure near the probe.

[0112] The mesoporous metal probe (AFM probe) of the present invention will further expand the research field that combines mesoporous metal materials with AFM.

[0113] (Figure 9) Figure 9 shows the fields of ultra-precision nanofabrication and ultra-high sensitivity nanomeasurement that can be opened up by the mesoporous metal probe (AFM probe) of the present invention. The present invention makes it possible to apply combinations of metal films that serve as the base for deposition on AFM and mesoporous metal materials (mesoporous films and mesoporous nanoparticles), enabling the creation of new functions.

[0114] The mesoporous metal materials described in this invention, in addition to the Au used in the examples, can lead to techniques for forming / modifying mesoporous films and mesoporous nanoparticles of various inorganic materials (platinum (Pt), silver (Ag), gold-silver (Au-Ag) alloys, etc.) and oxide semiconductors (TiO2, etc.).

[0115] For example, by processing the surface of a probe with mesoporous Pt, the catalytic reaction can be highly activated, enabling chemically localized nanofabrication in the reaction solution. The present invention can be applied to ultra-precision nanofabrication such as localized nanofabrication using chemical catalytic reactions with Pt, and ultra-minimally invasive perforation of cell membranes using photocatalytic activity with TiO2. The present invention can be applied to high-sensitivity Raman spectroscopy measurements using Au, Ag, or Au-Ag alloys.

[0116] Probes used in integrated atomic force microscope (AFM) and Raman spectroscopy systems (manufactured by HORIBA, Oxford Instruments, BRUKER, etc.) are consumables and therefore have high business potential.

[0117] The mesoporous metal probe (mesoporous Au probe) of the present invention is easier to mass-produce by electroplating compared to gold nanoparticle-modified AFM probes that generate highly sensitive plasmon resonance. Because the concentration of gold ions in the solution is low and the electroplating time required for fabrication is short, the mesoporous metal probe of the present invention can be manufactured at low cost. With the increasing demand for nano-level measurement technology and the growth of the AFM field, the mesoporous AFM probe of the present invention is useful in the AFM field.

Claims

1. A probe having a metal with a mesoporous structure on its surface, wherein (A) the back surface has a structure in which the substrate is exposed, or (B) the back surface has a structure in which the metal with the mesoporous structure is absent.

2. The probe according to claim 1, wherein the surface is at least a portion of the surface of the tip of the probe.

3. The probe according to claim 1, wherein the back surface has an exposed thermally oxidized SiO2 surface and forms an insulating film.

4. The probe according to claim 1, wherein the metal is composed of at least one element selected from the group consisting of gold, silver, and copper.

5. The probe according to claim 1, wherein the probe has the mesoporous structure on the surface of its tip, the mesoporous structure is a three-dimensional nanostructure having pores with a diameter of 2 nm to 50 nm, and the tip has a structure that can puncture a cell membrane.

6. The probe according to claim 1, wherein the tip of the probe has the shape of a triangular pyramid, a square pyramid, or a cone, and the tip has a structure that can puncture a cell membrane.

7. The probe according to claim 1, wherein the probe has a tip diameter of 5 nm to 300 nm, and the tip has a structure that can puncture a cell membrane.

8. The probe according to claim 1, wherein the probe is a probe used for Raman spectroscopy and / or a probe used for shape measurement by an atomic force microscope.

9. A method for manufacturing a probe, comprising: electroplating the surface of the probe in an electrolyte containing a surfactant and metal ions by an electroplating method; reducing and depositing the metal on the surface of the probe to form a metal having a mesoporous structure on the surface of the probe.

Images