JP2010054336A - Discrimination method of microbody having element thin film existing on surface - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To discriminate the kind of a microbody whose surface is covered with two or more layers of thin films by measuring the amount of reflected electrons in electron beam inspection. <P>SOLUTION: The microbody surface is covered with two or more layers of thin films of various materials. In this case, the thin films are constituted of a reference layer used for immobilization of biomolecules using a common material and a variable layer used for discrimination using a different material. When constituting the thin films, the this films are manufactured, while strictly controlling the film thickness by using a resistance heating type deposition device or the like. The manufactured microbody is irradiated with an electron beam. A proper incident electron acceleration voltage is selected so that incident electrons reach the variable layer. The kind of the material of the microbody variable layer is discriminated from a difference of brightness of a reflected electron image acquired from the microbody by electron beam irradiation. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an inspection method for specifying a micro object whose surface is coated with a metal, a transition metal, a semiconductor, or the like.

  The characteristics and state of individual cells are significantly reflected in the amount and distribution of proteins present on the surface of the cells and mRNA present in the cells. In order to examine the expression level and distribution of these “target biomolecules” in detail, a minute structure is used as a label for the target molecule. The present inventors have proposed a method for producing microparticles that can be used for labeling a target biomolecule in JP-A-11-001703 “Preparation Method of Ultrafine Particles”. In addition, Japanese Patent Application Laid-Open No. 2006-153826 “Biological material labeling method and biological material inspection method” proposed a target biomolecule inspection method using fine particles as labels.

In Japanese Patent Laid-Open No. 11-001703, polystyrene spheres are dispersed in a single layer on a flat substrate, and metal or semiconductor is vapor-deposited so that the metal or semiconductor atoms that have traveled straight from the vapor deposition source are adsorbed only to the polystyrene sphere vapor deposition source side. A method of forming fine particles is disclosed.
JP-A-11-001703 JP 2006-153826 A

  However, if it is desired to detect by labeling multiple types of target biomolecules present in the cell at the same time, fine particles of different materials must be used as labels at the same time. No specific solution or method has been proposed for how to fix it.

  In addition, for example, when two or more layers of elements are vapor-deposited on a fine particle, a solution and a method regarding how to specify the kind of fine particle have not been proposed so far.

  Accordingly, there is a demand for development of a method for identifying the type of a micro object when there are a plurality of micro structures containing fine particles whose surfaces are coated with two or more layers in various materials.

  In view of the above situation, the present invention provides a method for identifying a micro object covered with a layered structure including one or more metals, transition metals, or semiconductors described below, a micro object used in such a method, A kit comprising such a microbody and a method for producing such a microbody are provided.

(Method of identifying minute bodies)
(1) A method for identifying a micro object covered with a layered structure including one or more metals, transition metals, or semiconductors,
Irradiating the microscopic object with an electron beam,
A step of measuring the amount of reflected electrons from the layered structure, and a step of identifying the micro object by the amount of reflected electrons
Including a method.

(Method for identifying target molecules using microscopic bodies)
(2) A method of using one or two or more kinds of microparticles coated with a layered structure containing one or more kinds of metals, transition metals, or semiconductors as a label for one or more kinds of target molecules Because
Immobilizing the microparticles to the target molecule;
Irradiating the microscopic object with an electron beam,
A step of measuring the amount of reflected electrons from the layered structure, and a step of identifying the micro object by the amount of reflected electrons
Including a method.

(3) In the above layered structure, by changing the number of layers, the kind of elements constituting each layer and / or the thickness of each layer, or a combination thereof for each layered structure, for each micro object having the above layered structure The method according to (1) or (2) above, wherein the amount of reflected electrons is different, and thereby one minute body can be distinguished from another minute body based on the amount of reflected electrons.
(4) Any of the above (1) to (3), wherein the layered structure is composed of one or more kinds of metal, transition metal, or semiconductor thin film laminated on the surface of the minute body. The method described in 1.
(5) The method of (4) above, wherein the thickness of each of the thin films ranges from about 1 nm to about 1 micron.
(6) The method according to (5) above, wherein the thin film is formed by resistance heating vapor deposition, chemical vapor deposition, or physical vapor deposition.
(7) Fine particles coated with a layered structure containing one or more metals, transition metals, or semiconductors, block-shaped structures, microstructures grown with crystals, biomolecules, or organic The method according to any one of (1) to (6) above, which is a molecule.
(8) The method according to (7) above, wherein the size of the microparticles is in the range of about 5 nm to about 100 microns.
(9) The step of irradiating the electron beam comprises
By appropriately setting or changing the accelerating voltage of the electron beam to be irradiated, the reach distance of the electron beam to the layered structure in the interlayer direction is appropriately set or changed, whereby the one or more kinds of metal Enabling detection of reflected electrons from a desired layer of transition metal, or semiconductor,
The step of identifying the micro body by the amount of reflected electrons,
Identifying the micro object according to the characteristics of the amount of reflected electrons from a predetermined layer of the layered structure or the characteristics of the amount of reflected electrons from a plurality of layers of the layered structure,
The method according to any one of (1) to (8) above.
(10) The step of irradiating the electron beam comprises
Setting or accelerating the electron beam acceleration voltage to a predetermined value within a range of about 0.1 kV to about 30 kV,
The method according to (9) above.
(11) The layered structure includes one or more reference layers and one or more variable layers.
The reference layer is composed of elements common to other layered structures,
The variable layer is composed of different elements or combinations of different elements that can be distinguished from other layered structures.
The method according to any one of (1) to (10) above.
(12) The reference layer is formed on the outermost surface of the layered structure, the variable layer is formed inside the reference layer, and a target molecule can be immobilized on the reference layer. The method described in 1.

(Micro body)
(13) A minute body covered with a layered structure including one or more metals, transition metals, or semiconductors, and based on the amount of reflected electrons of an electron beam irradiated to the minute body A micro object for use in a method for identifying a micro object.
(14) The layered structure includes one or more reference layers and one or more variable layers,
The reference layer is composed of elements common to other layered structures,
The variable layer is composed of different elements or combinations of different elements that can be distinguished from other layered structures.
The microbody according to (13) above.
(15) The reference layer is formed on the outermost surface of the layered structure, the variable layer is formed inside the reference layer, and a target molecule can be immobilized on the reference layer. The microscopic object described in 1.
(16) Fine particles covered with a layered structure containing one or more metals, transition metals, or semiconductors, block-like structures, crystal-grown microstructures, biomolecules, or organic The microbody according to any one of (13) to (15), which is a molecule.
(17) The microscopic body according to any one of (13) to (16), which has a size ranging from about 5 nm to about 100 microns.
(18) The microscopic body according to any one of (13) to (17), wherein the layered structure includes a thin film of the one or more metals, transition metal, or semiconductor.
(19) The microscopic object according to (18), wherein each thin film has a thickness in the range of about 1 nm to about 1 micron and is strictly controlled in angstrom units.
(20) The microbody according to any one of (13) to (19) for use in the method according to any one of (1) to (12).

(Kit containing micro objects)
(21) A kit including one or two or more kinds of minute bodies covered with a layered structure including one or more kinds of metals, transition metals, or semiconductors, and the minute objects are irradiated A micromolecule kit for identifying a target molecule for use in a method for identifying the microparticle based on the amount of reflected electrons of an electron beam.
(22) A micromolecule kit for identifying target molecules, comprising one or more micromolecules according to any one of (13) to (20).
(23) The micromolecule kit for target molecule identification according to (21) or (22), further including an instruction for use.

(Production method of micro object)
(24) The method for producing a microbody according to any one of (13) to (20),
A method comprising a step of sequentially depositing an element including one or more kinds of metals, transition metals, or semiconductors on a surface of a micro object to form a layered structure of a thin film of the element.
(25) The method according to (24), wherein the thin film is formed by resistance heating vapor deposition, chemical vapor deposition, or physical vapor deposition.

  In the present invention, reflected electrons generated when an electron beam is irradiated to the minute body are used in order to identify the type of the minute body whose surface is covered with two or more layers of various materials.

  In particular, by appropriately changing the acceleration voltage of electrons from the low voltage side to the high voltage side, when there are two or more thin films, a reflected electron image reflecting information on the material on the deeper layer side can be obtained from the surface. By using this method, the type of micro object can be identified.

  Therefore, the micro body is used to identify the type, and is used to fix the internal variable layer composed of various materials and the probe biomolecule, or to maintain the structure of the micro body stably. It can be made to be composed of a reference layer on the surface composed of a unified material.

  The brightness of the reflected electrons obtained when the electron beam is irradiated depends on the thickness of the thin film in addition to the type of material constituting the thin film, but according to the present invention, the film thickness is strictly measured in angstrom units. Controlled microscopic objects can be produced.

  According to the present invention, microscopic bodies composed of various types of materials can be easily identified, so that a plurality of target biomolecules of a cell can be labeled simultaneously by using microscopic bodies with different materials as a set. It can be used when. Further, by using the reference layer for immobilizing the probe biomolecule and the variable layer for identifying the type of the minute object, it is possible to produce a large number of minute objects that are easy to fix and identify the molecule.

1. Method for distinguishing micro-particles covered with a layered structure including one or more metals, transition metals, or semiconductors In one embodiment, the present invention is directed to one or more metals, transition metals. Or a method for identifying microscopic bodies coated with a layered structure comprising a semiconductor. This method
A process of irradiating a microscopic object with an electron beam,
A step of measuring the amount of reflected electrons from the layered structure, and a step of identifying a micro object by the amount of reflected electrons,
including.

Furthermore, the present invention provides, in one embodiment, one or more kinds of one or more kinds of microscopic bodies coated with a layered structure containing one or more kinds of metals, transition metals, or semiconductors. A method for use as a target molecule label is provided. This method
Immobilizing a microbody on a target molecule;
A process of irradiating a microscopic object with an electron beam,
A step of measuring the amount of reflected electrons from the layered structure, and a step of identifying a micro object by the amount of reflected electrons,
including.

In this specification, the term “metal” simply refers to a metal of a typical element. “Typical elements” are elements of Groups 1, 2 and 12 to 18 in the periodic table, and are classifications of elements composed of all non-metals and some metals. As the “metal” used in the present invention, for example, metals such as Al, Ga, Ge, As, In, Sn, Sb, Tl, Pb, and Bi are preferable.
In the present specification, the “transition metal” means a transition element, which means an element from Group 3 to Group 11 of the periodic table. The “transition metal” used in the present invention is preferably a transition metal from 21 to 79 excluding atomic number 43.
In this specification, the term “semiconductor” has a meaning commonly used in the art (“a substance having an electrical conductivity σ at room temperature of about 10 ³ to 10 ⁻¹⁰ S / cm between a metal and an insulator”). (Iwanami Physical and Chemical Dictionary 5th edition, 1998 Iwanami Shoten)). As the “semiconductor” used in the present invention, for example, Si, Se, Te and the like are preferable.

  In the present specification, the “microbody” has a size (or particle size) of about 0.1 nm to about 1 mm, preferably about 1 nm to about 500 μm, more preferably about 5 nm to about 100 μm, most preferably about It refers to fine particles of 5 nm to 1 μm, regardless of the material. Examples of preferable microscopic materials used in the present invention include polystyrene, glass, silicon microparticles, glass or silicon block-like structures, microscopically grown microstructures such as zinc oxide and titanium oxide, and biomolecules. (Example: protein crystal) or organic molecule (eg, liposome).

  In the present specification, the “layered structure” means a structure in which thin layers formed of one or more metals, transition metals, or semiconductors are stacked. The thickness of each layer typically ranges from about 1 nm to about 1 μm, more preferably from about 1 nm to about 500 nm, most preferably from about 1 nm to about 100 nm, but limited to these ranges However, the lower limit can be appropriately set in accordance with the purpose within an arbitrary range from about 0.1 nm to the upper limit of about 10 μm.

  A “layered structure” can be made on any suitable surface by methods well known in the art. More specifically, for example, by using a resistance heating vapor deposition method, a chemical vapor deposition method, a physical vapor deposition method, or the like, a thin film of one kind or two or more kinds of metals, transition metals, or semiconductors, It can be produced by vapor deposition on a suitable surface. Those skilled in the art can understand that the thickness of the element to be deposited can be appropriately adjusted by these methods.

  The “layered structure” preferably comprises one or more reference layers and one or more variable layers. Here, “reference layer” means a layer composed of elements common to other layered structures, and “variable layer” means a layer composed of different elements that can be distinguished from other layered structures (or A plurality of layers composed of a combination of different elements that can be distinguished from other layered structures).

  Preferably, the “reference layer” is formed on the outermost surface of the layered structure, and the variable layer is formed inside the reference layer. By adopting such a form, the target molecule can be immobilized on the outermost reference layer. Preferred reference layer elements for binding to the target molecule include, for example, gold, silver, titanium, nickel and the like.

  In the present specification, the “target molecule” means a molecule as a target to be labeled with the microbody of the present invention that can bind to the microbody of the present invention. Examples of the “target molecule” include DNA, RNA, protein, sugar chain and the like present in a biological sample. The target molecule can be immobilized on the reference layer by, for example, immobilizing DNA, RNA, or an antibody that selectively binds to the target molecule on the surface of the reference layer and selectively reacting with the molecule. The method of immobilizing DNA, RNA, and antibodies on the surface of the reference layer is, for example, by reacting DNA or RNA with a thiol group at the terminal or a cysteine residue of a protein against the reference layer surface made of gold. Can be fixed.

  In this way, the target molecules can be uniformly bound to the microscopic body covered with the layered structure. Further, another reference layer may be provided on the innermost side of the layered structure. In this case, the innermost reference layer can be used to stabilize the structure of the minute body or to evaluate the depth of penetration of the electron beam into the minute body.

  Further, the presence of the “variable layer” makes it easy to distinguish one “fine particle” covered with such a layered structure from other “fine particles”. When using the microparticles of the present invention as a labeling tag, if a large number of the above-mentioned “microparticles” that can be distinguished from each other are required (for example, when there are a large number of different target molecules), variations in the “variable layer” are provided. By providing appropriately, a large number of labeling microparticles that can be distinguished from other microparticles can be produced. Giving variations to the variable layer means changing the number of layers constituting the variable layer, changing the type and / or combination of elements in each layer, changing the thickness of the variable layer, or these It can be performed by a combination or the like.

  In the method for identifying a micro object according to the present invention, the micro object covered with the layered structure as described above is irradiated with an electron beam, the amount of reflected electrons from the layered structure is measured, and the difference in the amount of reflected electrons is determined. Identify each particulate.

  Here, “electron beam” refers to a beam emitted from an electron beam source (electron gun). Typically, the electron beam source is incorporated into a scanning electron microscope. When an electron beam emitted from an electron beam source enters the layered structure, secondary electrons, reflected electrons, and the like are emitted. These emitted signals are detected by a detector and displayed after amplification and modulation. The signal processing result is displayed as an image on the display and observed.

  The amount of reflected electrons depends on the material where the incident electrons collide, and a heavier element produces more reflected electrons. Since the amount of reflected electrons is reflected in the brightness of the reflected electron image, a minute object made of a heavy material becomes brighter. Therefore, by obtaining the brightness of the reflected electrons according to the type of element, the element can be identified based on the amount of reflected electrons (or brightness).

  On the other hand, the electrons that make up the incident electron beam are accelerated by applying an acceleration voltage. However, as the larger acceleration voltage is applied, the incident electrons have a larger energy, so that they enter the deeper part from the sample surface. . By appropriately setting or changing the acceleration voltage of the electron beam, the reach distance of the electron beam to the layered structure in the interlayer direction can be set or changed as appropriate. The electron beam accelerating voltage is typically set to a predetermined value in the range of about 0.1 kV to about 30 kV, or can be varied within that range, but the equipment used (eg, scanning type) Depending on the electron microscope, the range is not limited to the above.

  By setting the variable layer to one layer or two or more layers and appropriately changing the types of elements constituting each of the layers, various types of layered structures can be produced. In such a layered structure, when the amount of reflected electrons is measured while appropriately setting or changing the reach distance of the electron beam to the layered structure in the interlayer direction, the brightness of reflected electrons (or the amount of reflected electrons) in each different layer direction, Alternatively, since the pattern of luminance (or amount of reflected electrons) is shown, the layered structure can be distinguished from other layered structures by the unique luminance or luminance pattern.

2. Microbody of the Invention The present invention also provides, in another embodiment, a microbody coated with a layered structure comprising one or more metals, transition metals, or semiconductors. This minute body can be used in a method of identifying the minute body based on the amount of reflected electrons of the electron beam irradiated to the minute body.

  In the micro body of the present invention, the layered structure typically includes one or more reference layers and one or more variable layers. Here, the reference layer is composed of elements common to other layered structures, and the variable layer is composed of different elements or combinations of different elements that can be distinguished from other layered structures. The description of the “layered structure”, “reference layer”, and “variable layer” is as already described in the description of the fine particle identification method of the present invention.

  Typically, the microbody of the present invention is used in the above-described microbody identification method or the method of using the microbody as a label (tag).

3. In another embodiment of the present invention , a kit containing one or two or more kinds of microparticles coated with a layered structure containing one or more kinds of metals, transition metals, or semiconductors. A microbody kit for identifying a target molecule is provided. This kit can be used in a method of identifying a minute body based on the amount of reflected electrons of an electron beam irradiated to the minute body. The kit of the present invention can contain one or more of the microparticles of the present invention. Furthermore, the kit of the present invention may contain instructions for use. The kit of the present invention is typically used in the above-described microbody identification method of the present invention or a method of using the microbody as a label (tag).

4). In yet another embodiment the production method the present invention the micro of the present invention to provide a method for producing a fine body of the present invention. This method is an element containing one or more metals, transition metals, or semiconductors on the surface of a “microscopic object” made of any material (eg, polystyrene beads, glass beads, silicon pieces, zinc oxide crystals, etc.). Are sequentially deposited to form a layered structure of a thin film of the above elements. This thin film is typically formed by resistance heating vapor deposition, chemical vapor deposition, or physical vapor deposition. Among these, a method using a resistance heating type vapor deposition apparatus that is easy to handle and capable of strictly controlling the film thickness in angstrom units is preferable.

  Hereinafter, more specific examples of the present invention will be described with reference to the drawings, but the present invention is not limited to these examples.

FIG. 1 is a schematic view of a micro object having a thin film formed on the surface to be identified in the present invention. Here, five types of micro objects 1, 2, 3, 4, 5 having different materials are shown as examples. The reference body 1 ₁ , 1 ₄ , 2 ₁ , 2 ₄ , 3 ₁ , 3 ₄ , 4 ₁ , 4 made of metal, transition metal and semiconductor having a size of 5 to 100 microns and a thickness of 1 to 1 micron. ₄ , 5 ₁ , 5 ₄ , and variable layers 1 ₂ , 1 ₃ , 2 ₂ , 2 ₃ , 3 ₂ , 3 ₃ , 4 ₂ , 4 ₃ , 5 ₂ , 5 ₃ . The same material is used for the reference layers 1 ₁ , 2 ₁ , 3 ₁ , 4 ₁ , 5 ₁ . The same applies to 1 ₄ , 2 ₄ , 3 ₄ , 4 ₄ , 5 ₄ . The variable layers 1 ₂ , 1 ₃ , 2 ₂ , 2 ₃ , 3 ₂ , 3 ₃ , 4 ₂ , 4 ₃ , 5 ₂ , and 5 ₃ are made of different materials, or 1 ₂ , 1 _3, and 5 _{2, respectively.} two types of material as 5 ₃ form a thin film in a different order. Here, an example of a micro object composed of two reference layers and two variable layers has been shown, but the number of layers may be selected in the range of about 1 to 10 layers depending on the application. Each microbody labels a different target biomolecule 6.

  FIG. 2 is a schematic view of a method for forming a thin film on a microscopic object. As a typical example, a process of forming a thin film of an arbitrary metal, transition metal or semiconductor on the surface of the micro object 9 by a resistance heating type vapor deposition apparatus is shown. Examples of the material of the micro body 9 include 5 micron to 100 micron living bodies such as fine particles such as polystyrene beads, block-like structures such as silicon pieces, crystal-grown micro structures such as zinc oxide, protein crystals and liposomes. It is better to use molecules and organic molecules. Here, an example is shown in which a thin film is formed by a resistance heating vapor deposition apparatus that is easy to handle and the film thickness can be controlled in angstrom units. However, any metal, transition metal or transition metal or chemical vapor deposition method is used. A semiconductor thin film may be formed. When two or more kinds of elements are vapor-deposited, it is preferable to prepare two or more containers like the vapor deposition sources 11 and 12 in FIG. 2 and select and use the material to be vapor-deposited by switching the wiring.

As shown in FIG. 2, first, the micro object 9 is fixed in advance on a support substrate 8 made of glass, silicon, plastic, or the like, and is placed in the chamber of the resistance heating evaporation apparatus so as to face the evaporation sources 11 and 12. set. The degree of vacuum in the chamber is, for example, 5 × 10 ⁻⁵ Pascal, and the temperature in the chamber is room temperature. A shutter 10 is provided between the minute body 9 and the vapor deposition sources 11 and 12. Evaporation source 11 (hereinafter, the deposition source 12 is the same) consists of the evaporation source container ₁₁₁ and the heating resistor 11 _2. The shutter 10 can be moved as indicated by the arrow displayed on the left side of the drawing. When the shutter 10 covers the entire surface of the microbody-immobilized support substrate 8, vapor deposition on the microbody 9 is prevented, and the shutter 10 When the microbody-immobilized support substrate 8 is exposed to the vapor deposition source 11 by the movement, vapor deposition onto the microbody 9 is performed. The evaporation source container 11 ₁ is deposited on the surface of the minute body 9, a metal serving as a reference layer and deformable layer, a transition metal or a semiconductor is placed. Heating resistor 11 ₂ heats the element placed in the evaporation source container ₁₁₁ is a resistor to vaporize.

  Here, the process of forming a thin film by performing vapor deposition on a micro object fixed to the substrate is shown, but in addition to this, elements are vapor-deposited in advance on a flat substrate, and the surface is cut and cut out by etching. A method of forming a body, a part of a flat substrate is covered with a mask, a region that is not exposed to an element is formed, a thin film is formed, and a part is cut out to form a microscopic body, a fine particle or a microstructure There is a method of attaching finer particles and structures to the surface of the film.

Examples of elements that can be used as vapor deposition sources (elements for forming a thin film) in the present invention are listed in the periodic table as follows.
(1) transition metals up to 79 excluding atomic number 43,
(2) Metals with atomic numbers 13, 31, 32, 33, 49, 50, 51, 81, 82, and 83, and (3) Semiconductors with atomic numbers 14, 34, and 52.

By performing the process of FIG. 2 and forming a thin film on the surface of the micro object, a micro object covered with the multilayered thin film shown in FIG. 1 is produced. By heating in the order of the heating resistors 11 ₂ and 12 ₂ , a two-layer thin film is formed on the surface of the micro object 9. The thickness of the thin film is controlled in units of angstroms depending on the current and voltage values applied to the heating resistors 11 ₂ and 12 ₂ and the time during which the shutter 10 is released during vapor deposition. When forming a thin film having three or more layers, it is preferable to increase the number of vapor deposition sources or to sequentially replace the materials in the vapor deposition source containers 11 ₁ and 12 ₁ . The formed thin film is used as a reference layer or a variable layer. The micro body 9 that forms the base for forming the thin film is treated as one of the reference layers in the micro body after the thin film is formed.

FIG. 3 is a schematic diagram for explaining the procedure for identifying the type of micro object by electron beam inspection. Here, an example is shown in which a sample 7 labeled with five kinds of microscopic objects 1, 2, 3, 4, and 5 is placed on a sample stage 14 and observed with a scanning electron microscope 13.
When electrons of the electron beam 13-2 emitted from the electron gun 13-1 collide with the minute bodies 1, 2, 3, 4 and 5, secondary electrons 13-3 and reflected electrons 13-4 are emitted from the minute bodies. Is done. The secondary electrons are captured by the detector 13-5 and the reflected electrons are captured by the detector 13-6. Based on the secondary electrons detected by the detector 13-5, a secondary electron beam image is obtained, and the position and size of the micro object are specified. Based on the reflected electrons detected by the detector 13-6, a reflected electron beam image is obtained, and the position, size and material of the micro object are specified. Since the secondary electron beam image can be obtained with higher resolution and higher speed than the reflected electron beam image, it is preferable to appropriately use the reflected electron image together with the reflected electron image when examining the position and size of the micro object in detail.

  FIG. 4 is a schematic diagram for explaining the principle of identifying the type of micro object by the reflected electron image. When the incident electron beam 13-2 is irradiated to the microscopic object, the reflected electron 13-4 is emitted from the microscopic object, and the amount depends on the material of the portion where the incident electron collides, and the heavier element, the more reflected electrons. Occurs. Since the amount of reflected electrons is reflected in the brightness of the reflected electron image, a minute object made of a heavy material becomes brighter. On the other hand, the electrons that make up the incident electron beam are accelerated by applying an acceleration voltage. However, as the larger acceleration voltage is applied, the incident electrons have a larger energy, so that they enter the deeper part from the sample surface. .

The micro objects 1, 2, 3, 4, and 5 are composed of a reference layer on the surface and an internal variable layer. When the acceleration voltage applied to the incident electron beam 13-2 is low, as shown in FIG. 4 (a), the incident electrons are the reference layers 1 ₁ , 2 ₁ , 3 ₁ , 4 ₁ , 5 near the surface where the materials are unified. _Since only ₁ can be reached, the reflected electron image luminances of the micro objects 1, 2, 3, 4, and 5 are all the same. However, when applying a higher acceleration voltage, to reach 4 incident electrons, as shown in (b) the variable layer ₁ 2 different materials, _{respectively,} 2 _2, 3 _2, 4 2, _{5 2,} microbodies 1, 2, 3, 4, and 5 have different reflected electron image luminances. That is, the type of the micro object can be specified from the reflected electron image luminance.

When a higher acceleration voltage is applied, the incident electrons reach the deeper variable layers 1 ₃ , 2 ₃ , 3 ₃ , 4 ₃ , and 5 ₃ , so that the microscopic objects 1, 2, ₃ , 4, and 5 are reflected differently. Electronic image brightness. By utilizing this, the same material 1 ₂ and 5 _3, and 1 ₃ and 5 ₂ of the thin film are stacked in a different order, it is possible to identify a small body 1 and the minute body 5.

  As an example, when a gold and silver thin film of 20 nm is configured as a variable layer on the surface of a micro object and 20 nm of copper is stacked as a reference layer thereon, the luminance is the same when the incident electron acceleration voltage is low, but the acceleration voltage When the value exceeds 5 kV, the reflected electron brightness of the microscopic structure in which copper is laminated on gold becomes brighter than that of the microscopic structure in which copper is laminated on silver. Since the relationship between the penetration depth of the incident electrons and the acceleration voltage depends on the type of material constituting the micro object, it is preferable to select an appropriate value for the acceleration voltage in the range of 0.1 to 30 kV.

  FIG. 5 is a schematic diagram showing the relationship between the brightness of reflected electrons obtained from a microscopic object having a thin film laminated on the surface, the atomic number of the material constituting the thin film, and the thickness of the thin film. As shown in FIG. 5A, the greater the atomic number of the thin film material, the brighter the reflected electron image, but the relationship is not linear, and the heavier elements are more difficult to identify. Therefore, when using a set of microscopic objects of various materials as a label, it is necessary to consider the combination of materials used simultaneously. For example, five kinds of elements such as aluminum, chromium, copper, silver, and gold can be used at the same time because their atomic numbers are different and the reflected electron luminance is clearly different.

  On the other hand, as shown in FIG. 5 (b), the reflected electron luminance becomes brighter as the thickness of the thin film covering the micro object increases, and the relationship is linear. When the acceleration voltage of incident electrons is low, the depth of penetration of the incident electrons into the sample becomes shallow, so that a thin film with a certain thickness or more cannot be identified, and the tendency as shown by the dotted line in FIG. Therefore, it is preferable to select an appropriate value in the range of 0.1 to 30 kV for the acceleration voltage according to the type of micro object to be used. For example, if the material of the thin film that covers the microscopic object is gold, the linearity is maintained up to the film thickness of 20 nm at the acceleration voltage of 10 kV, but the linearity is lost in the region of the film thickness of 10 nm or more when it is 5 kV .

  Since the relationship between the thickness of the thin film and the reflected electron luminance is as described above, the thickness of the thin film covering the micro object needs to be strictly controlled in angstrom units. By using a resistance heating type vapor deposition apparatus, it is possible to easily produce a microscopic body whose thickness is strictly controlled.

  As described above, the present invention includes a reference layer that has a common material on the surface and is used for immobilizing probe biomolecules, etc., and a variable layer that is composed of various materials and is used to identify the type. For a micro object formed of two or more layers, an appropriate incident electron beam acceleration voltage is selected, a reflected electron image reflecting the variable layer material information is obtained, and the brightness is compared to identify the type of the micro object. be able to. By utilizing this, it is possible to produce a large amount of various types of microscopic bodies that can be easily immobilized and identified with probe biomolecules. It can be labeled and can be discriminated and measured.

It is a schematic diagram which shows the example of the various target molecules couple | bonded with the various micro bodies of this invention in which the layer of thin films, such as a metal, was formed in the surface. It is a schematic diagram which shows the process of forming a thin film on the surface of a micro object using a resistance heating type vapor deposition apparatus. It is a schematic diagram which shows the procedure which identifies the kind of micro object by an electron beam test | inspection. (A) is a schematic diagram showing a state of reflected electrons emitted when electrons having a low acceleration voltage are incident on a micro object having a thin film formed on the surface. (B) is a schematic diagram showing a state of reflected electrons emitted when the acceleration voltage is high. (A) is a figure which shows the relationship between the brightness | luminance of the reflected electron obtained from a micro object, and the atomic number of the raw material which coat | covers a micro object. (B) is a figure which shows the relationship between the brightness | luminance of the reflected electron obtained from a micro object, and the thickness of the thin film which coat | covers a micro object.

Explanation of symbols

1,2,3,4,5 ... microbodies coated with a thin film of _multi-layer, 1 _1, 2 _1, 3 1, _{4 1,} 5 1 _... reference layer _1,1 2 made of the same _{material, 2} 2, 3 ₂ , 4 ₂ , 5 ₂ ... variable layers 1, 1 ₃ , 2 ₃ , 3 ₃ , 4 ₃ , 5 ₃ ... variable layers 2, 1 ₄ , 2 ₄ , 3 ₄ , 4 ₄ , 5 of different materials ₄ ... Reference layer 2 of the same material, 6 ... Different types of target biomolecules, 7 ... Organic substances such as cells, 8 ... Support substrate for fixing the minute bodies, 9 ... Minute bodies, 10 ... Shutters, 11 ₁ ... Deposition source Container 1, 11 ₂ ... Heating resistor ₁ , 12 ₁ ... Evaporation source container 2, 12 ₂ ... Heating resistor ₂ , 13-1 .. Electron gun, 13-2. , 13-4 ... backscattered electrons, 13-5 ... secondary electron detector, 13-6 ... backscattered electron detector, 14 ... scanning electron microscope sample stage.

Claims (25)

A method for identifying a microscopic object coated with a layered structure comprising one or more metals, transition metals, or semiconductors, comprising:
Irradiating the minute body with an electron beam,
Measuring the amount of reflected electrons from the layered structure; and identifying the minute body by the amount of reflected electrons;
Including a method. A method of using one or more kinds of microparticles coated with a layered structure containing one or more kinds of metals, transition metals, or semiconductors as a label for one or more kinds of target molecules. ,
Immobilizing the microbody to the target molecule;
Irradiating the minute body with an electron beam,
Measuring the amount of reflected electrons from the layered structure; and identifying the minute body by the amount of reflected electrons;
Including a method.   In the layered structure, the number of layers, the kind of elements constituting each layer and / or the thickness of each layer, or a combination thereof is changed for each layered structure, whereby different reflected electrons are provided for each micro object having the layered structure. 3. The method according to claim 1 or 2, wherein a quantity is indicated so that one micro object can be distinguished from another micro object based on the amount of reflected electrons.   The method according to any one of claims 1 to 3, wherein the layered structure is composed of one or more kinds of metal, transition metal, or semiconductor thin film laminated on the surface of the micro body.   The method of claim 4, wherein the thickness of each of the thin films ranges from about 1 nm to about 1 micron.   The method according to claim 5, wherein the thin film is formed by resistance heating vapor deposition, chemical vapor deposition, or physical vapor deposition.   The fine body is a fine particle, a block-like structure, a crystal-grown microstructure, a biomolecule, or an organic molecule that is coated with a layered structure including one or more metals, transition metals, or semiconductors. The method according to claim 1.   8. The method of claim 7, wherein the size of the microparticles ranges from about 5 nm to about 100 microns. Irradiating the electron beam,
By appropriately setting or changing the accelerating voltage of the electron beam to be irradiated, the reach distance in the interlayer direction of the electron beam to the layered structure is appropriately set or changed, and thereby the one or more kinds of metals Enabling detection of reflected electrons from a desired layer of transition metal, or semiconductor,
The step of identifying the micro object by the amount of reflected electrons,
Identifying the micro object according to a characteristic of the amount of reflected electrons from a predetermined layer of the layered structure or a characteristic of the amount of reflected electrons from a plurality of layers of the layered structure.
The method according to claim 1. Irradiating the electron beam,
Setting or changing the acceleration voltage of the electron beam to a predetermined value within a range of about 0.1 kV to about 30 kV,
The method of claim 9. The layered structure comprises one or more reference layers and one or more variable layers;
The reference layer is composed of elements common to other layered structures,
The variable layer is composed of different elements or combinations of different elements that can be distinguished from other layered structures,
The method according to claim 1.   The method according to claim 11, wherein the reference layer is formed on an outermost surface of the layered structure, the variable layer is formed inside the reference layer, and target molecules can be immobilized on the reference layer. .   A minute body covered with a layered structure including one or more metals, transition metals, or semiconductors, wherein the minute body is formed based on the amount of reflected electrons of an electron beam irradiated to the minute body. A micro object for use in a method of identification. The layered structure comprises one or more reference layers and one or more variable layers;
The reference layer is composed of elements common to other layered structures,
The variable layer is composed of different elements or combinations of different elements that can be distinguished from other layered structures,
The microbody according to claim 13.   The microscopic layer according to claim 14, wherein the reference layer is formed on the outermost surface of the layered structure, the variable layer is formed inside the reference layer, and target molecules can be immobilized on the reference layer. body.   The fine body is a fine particle, a block-like structure, a crystal-grown microstructure, a biomolecule, or an organic molecule that is coated with a layered structure including one or more metals, transition metals, or semiconductors. The microbody according to any one of claims 13 to 15.   17. A microbody according to any of claims 13 to 16, which is a size in the range of about 5 nm to about 100 microns.   The microscopic object according to any one of claims 13 to 17, wherein the layered structure is composed of the one or more kinds of metal, transition metal, or semiconductor thin film.   19. A microbody according to claim 18, wherein the thickness of each of the thin films is in the range of about 1 nm to about 1 micron and is tightly controlled in angstrom units.   The microbody according to any one of claims 13 to 19 for use in the method according to any one of claims 1 to 12.   A kit including one or two or more kinds of minute bodies coated with a layered structure including one or more kinds of metals, transition metals, or semiconductors, wherein the electron beam irradiated to the minute bodies A micromolecule kit for identifying a target molecule for use in a method for identifying the microparticle based on the amount of reflected electrons.   A micromolecule kit for identifying target molecules, comprising one or more micromolecules according to any one of claims 13 to 20.   23. The micromolecule kit for target molecule identification according to claim 21 or 22, further comprising instructions for use. It is a manufacturing method of the micro object according to any one of claims 13 to 20,
A method comprising the step of sequentially depositing one or more elements including a metal, a transition metal, or a semiconductor on the surface of a micro object to form a layered structure of a thin film of the element.   25. The method of claim 24, wherein the thin film is formed by resistance heating vapor deposition, chemical vapor deposition, or physical vapor deposition.