JP5046210B2 - Method for forming fine particles and method for inspecting biological material using the fine particles - Google Patents

Description

  The present invention relates to a method for forming fine particles of metal, semiconductor, etc. and a method for inspecting a biological material using the fine particles.

It is effective to investigate the expression status of genes in order to understand life phenomena and investigate the functions of genes. For this reason, the use of fine particles as a biomarker is performed. The inventors of the present application have proposed a method for producing fine particles that can be used as a biomarker, for example, in JP-A-11-001703 “Preparation method of ultrafine particles”. In addition, Japanese Patent Application Laid-Open No. 2006-153826 “Biological sample labeling and biological material labeling method and biological material inspection method” proposed a biological material inspection method using microparticles as a biological label.
JP-A-11-001703 JP 2006-153826 A

  In JP-A-11-001703, polystyrene spheres are dispersed in a single layer on a flat substrate, and a metal or semiconductor is deposited to adsorb metal or semiconductor atoms that have traveled straight from the evaporation source only on the polystyrene sphere deposition source side. A method of forming fine particles is disclosed.

  However, a method of dispersing fine particles such as polystyrene spheres on a flat substrate at a higher density, and a modified fine particle in which metal or semiconductor atoms are adsorbed only on the deposition source side of fine particles such as polystyrene spheres are peeled off from the substrate. Thus, there is no specific disclosure of a method for making individual modified fine particles.

  Therefore, disclosure of a more specific and effective method for forming modified fine particles is desired, including these methods.

  Furthermore, it is desired to improve an inspection method for biological substances using modified microparticles as a biomarker.

  In view of the above circumstances, the present invention provides a method for producing fine particles modified with transition metals, metals, or semiconductors, and a method for inspecting biological materials.

(1) preparing a substrate, a silicon substrate, or a glass substrate having a gold thin film formed on one surface;
Mixing polystyrene fine particles having a predetermined diameter, SiO ₂ fine particles, or TiO ₂ fine particles, an appropriate amount of pure water, and a material for suppressing electrostatic repulsion between the fine particles, on the substrate A step of preparing a fine particle suspension suspended so that the fine particles can be distributed at a high density;
Dropping the fine particle suspension onto the substrate to distribute the fine particles at a high density on the substrate;
Cleaning and removing excessive amounts of fine particles not adsorbed on the substrate, and drying the fine particles distributed on the substrate;
Depositing at least one layer of transition metal, metal or semiconductor on the fine particles distributed on the substrate;
A step of forming one layer of SiO ₂ by vapor deposition or sublimation on the fine particles on which at least one layer of the transition metal, metal or semiconductor is vapor-deposited;
A small amount of pure water or bovine serum albumin (BSA) as a protein in the pure water and a citrate buffer (standard as a buffer) on the fine particles fixed on the substrate and vapor-deposited. sodium citrate (SSC)) or phosphate buffer, and a step of adding a small amount of liquid to which sodium dodecyl sulfate (SDS) or tannic acid is added as a surfactant, And placing a material having a flat bottom surface on the one surface side to which the fine particles are fixed while applying ultrasonic waves to the other surface of the substrate so that a slight load is applied to the bottom surface. Separating the fine particles from the substrate by moving in a direction,
Of fine particles modified with transition metals, metals or semiconductors.
(2) The method for producing fine particles according to (1) above, wherein the material for suppressing electrostatic repulsion between the fine particles is 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride, NaCl, or KCl.
(3) The transition metal, metal or semiconductor is a transition metal up to 79 in the periodic table except atomic number 43, atomic number 13, 31, 32, 33, 49, 50, 51, 81, 82, or The method for producing fine particles according to the above (1), which is any one of the 83rd metal or the atomic number 14, 34, or 52th semiconductor.
(4) 0.01% to 10% of 3-aminopropyl trimethoxy silane, 3-aminopropyl triethoxy silane, or 3-aminopropyl diethoxy methyl silane is added to the fine particle suspension obtained by the step of separating the fine particles from the substrate. The method for producing fine particles according to the above (1), wherein an amino group is introduced into the fine particle surface by adding a silane coupling agent.
(5) A thiol group is added to the surface of the fine particles by adding 0.01% to 10% or less of a 3-mercaptopropyl trimethoxy silane silane coupling agent to the fine particle suspension obtained by separating the fine particles from the substrate. The method for producing fine particles according to (1), wherein
(6) A step of preparing a substrate, a silicon substrate, or a glass substrate on which a gold thin film is formed on one side,
Mixing polystyrene fine particles having a predetermined diameter, SiO ₂ fine particles, or TiO ₂ fine particles, an appropriate amount of pure water, and a material for suppressing electrostatic repulsion between the fine particles, on the substrate A step of preparing a fine particle suspension suspended so that the fine particles can be distributed at a high density;
Dropping the fine particle suspension onto the substrate to distribute the fine particles at a high density on the substrate;
Depositing at least one layer of transition metal, metal or semiconductor on the fine particles distributed on the substrate;
A solution of a silane coupling agent of 3-aminoprophyl trimethoxy silane, 3-aminoprophyl triethoxy silane, or 3-aminoprophyl diethoxy methyl silane around the substrate having the fine particles on which at least one layer of the transition metal, metal or semiconductor is deposited. A step of introducing an amino group on the surface of the fine particle by dripping a drop, sealing in a nitrogen or argon atmosphere container, and adsorbing a silane coupling agent molecule on the fine particle surface in a gas phase;
After dropping a small amount of pure water onto the fine particles fixed and deposited on the substrate, ultrasonic waves are applied to the other surface of the substrate, and the fine particles on the single layer are fixed. A step of separating the fine particles from the substrate by placing a material having a flat bottom surface on the surface so as to apply a load to the bottom surface and moving the material in an arbitrary direction;
Of fine particles modified with transition metals, metals or semiconductors.
(7) Instead of the 3-aminoprophyl trimethoxy silane, 3-aminoprophyl triethoxy silane, or 3-aminoprophyl diethoxy methyl silane silane coupling agent, a droplet of 3-mercaptopropyl trimethoxy silane silane coupling agent is dropped, and nitrogen is added. Or the manufacturing method of the microparticles | fine-particles of the said (6) description which introduce | transduces a thiol group into the said fine particle surface by sealing in an argon atmosphere container and making a silane coupling agent molecule | numerator adsorb | suck to the fine particle surface in a gaseous phase.
(8) After introducing amino groups on the surface of the fine particles, 0.1 mg to 1 g / mL of Sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] -propionamido) hexanoate (Sulfo-LC-SPDP), or Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC), etc. After activation with a cross-linking agent that crosslinks an amino group and a thiol group, at either 3 ′ or 5 ′ end The method for producing microparticles according to (1) above, wherein a biofunctional molecule is immobilized on the surface of the microparticle by reacting DNA or RNA into which a thiol group has been introduced, or a protein such as an antibody having a thiol group.
(9) After introducing a thiol group on the surface of the fine particles, 0.1 mg to 1 g / mL of Sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] -propionamido) hexanoate (Sulfo-LC-SPDP), or Sulfosuccinimidyl After activation with a cross-linking agent that crosslinks the thiol group and amino group, such as 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC), at either 3 ′ or 5 ′ end The method for producing microparticles according to (1) above, wherein a biofunctional molecule is immobilized on the surface of the microparticles by reacting amino group-introduced DNA or RNA, or an amino group-containing protein or the like.
(10) The method for producing fine particles according to the above (8) or (9), wherein the two-crosslinking agent is reacted with the fine particles after first reacting with a biofunctional molecule.
(11) After introducing amino groups on the surface of the fine particles, 0.1 mg to 1 g / mL of Sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] -propionamido) hexanoate (Sulfo-LC-SPDP), or Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC), etc. After activation with a cross-linking agent that crosslinks an amino group and a thiol group, at either 3 ′ or 5 ′ end The method for producing microparticles according to (6) above, wherein a biofunctional molecule is immobilized on the surface of the microparticle by reacting DNA or RNA into which a thiol group has been introduced, or a protein such as an antibody having a thiol group.
(12) After introducing a thiol group on the surface of the fine particles, 0.1 mg to 1 g / mL of Sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] -propionamido) hexanoate (Sulfo-LC-SPDP), or Sulfosuccinimidyl After activation with a cross-linking agent that crosslinks the thiol group and amino group, such as 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC), at either 3 ′ or 5 ′ end The method for producing microparticles according to (7) above, wherein a biofunctional molecule is immobilized on the surface of the microparticles by reacting amino group-introduced DNA or RNA and amino group-containing proteins.
(13) The method for producing fine particles according to the above (11) or (12), wherein the two-crosslinking cross-linking agent is reacted with the fine particles after first reacting with a biofunctional molecule.
(14) Using a fine particle made of polystyrene, a fine particle made of SiO ₂ , or a fine particle made of TiO _{2 having a} predetermined diameter, the surface of which is modified with a transition metal, metal, or semiconductor, and a probe fixing region on the surface of a chip to be observed A target molecule containing DNA, mRNA, or protein is bound to a number of probes fixed to the microparticle, and the target molecule is labeled via a biofunctional molecule on the surface of the microparticle, and the observation target Obtaining a reflected electron beam image by electron beam scanning of the fine particles obtained by simultaneously scanning the fine particles for comparison provided at a position separated from the surface of the probe fixing region of the chip with an electron beam,
A living body characterized in that a target molecule containing the DNA, mRNA, or protein is specified by determining a material that modifies the surface of the fine particle from the brightness of the reflected electron beam image of the labeled fine particle. Inspection method for substances.
(15) The material that modifies the surface of the fine particle is determined by comparing the reflected electron beam image of the comparative fine particle with the reflected electron beam image of the labeled fine particle, and the DNA, mRNA, or The biological material test method according to (14), wherein a target molecule containing a protein is specified.
(16) In addition to the reflected electron beam image obtained by electron beam scanning of the fine particles obtained by scanning with the electron beam, a secondary electron beam image is obtained from the secondary electrons obtained by scanning with the electron beam. The biological material inspection method according to (14), wherein the position of the fine particles in the line image is specified.
(17) In addition to the reflected electron beam image obtained by electron beam scanning of the fine particles obtained by scanning with the electron beam and the secondary electron beam image obtained by scanning with the electron beam, the electron beam is scanned. The method for inspecting a biological material according to (14) above, wherein an element spectrum image of a material that modifies the surface of the fine particles obtained is obtained, and a composition element of the fine particles is specified.
(18) An element mapping image of the material modifying the surface of the fine particles is obtained instead of the elemental spectrum image of the material modifying the surface of the fine particles obtained by scanning with the electron beam, and the fine particles The biological material test method according to (14), wherein the composition element is specified.

  In the present invention, gold is vapor-deposited with a predetermined thickness on the surface of a substrate on which fine particles are fixed in a single layer. Alternatively, a silicon substrate or a glass substrate is used as a substrate on which fine particles are fixed to a single layer. On the other hand, a particle fixing solution using a material for suppressing electrostatic repulsion between fine particles such as 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (commonly known as EDC), NaCl, or KCl is prepared. A fine particle is fixed to the substrate surface as a single layer by applying a fine particle suspension onto the substrate as a particle suspension.

  In order to peel the modified fine particles from the substrate, the ultrasonic wave is applied to the back surface of the substrate using an ultrasonic cleaning device or the like to promote the peeling.

  Furthermore, when the modified fine particle is modified with a biological functional molecule and used as a label for the inspection of the biological material, reflected electrons from the modified fine particle are used as an improvement of the inspection method of the biological material.

  According to the present invention, various metal-modified fine particles or semiconductor-modified fine particles can be easily formed. In addition, these modified fine particles can be modified with biological functional molecules to more effectively identify and detect biological substances.

  FIGS. 1A to 1E are conceptual diagrams illustrating a process of fixing a single layer of fine particles on a substrate in the first step of the modified fine particle production process of the present invention. In the present invention, a plastic substrate, a silicon substrate, or a glass substrate such as a slide glass can be used as the substrate. Here, it is assumed that a 35 mm plastic Petri dish that is inexpensive and easily available is used. To do. The basic particles are polystyrene particles or polystyrene particles containing a magnetic material. The size of these fine particles may be arbitrarily selected according to the purpose of use. For example, those having an appropriate diameter may be selected from those having a diameter of 10 nm to 150 μm.

  FIG. 1A is a plan view showing a state in which gold 2 is deposited on the bottom surface of a 35 mm plastic petri dish 1 with a thickness of, for example, 10 nm to 30 nm. By providing the gold 2 thin film, the polystyrene fine particles can be stably fixed to the bottom surface of the Petri dish 1. When a glass substrate is used, it can be used as it is. However, it is preferable to first deposit a chromium layer with a thickness of 20 nm and then deposit a gold 2 thin film with a thickness of 80 nm. In these cases, the thickness of the gold 2 thin film and the chromium layer is not limited to this value.

  FIG. 1 (b) shows a case in which a container 5 with a lid has polystyrene fine particles 3 having a predetermined diameter, an appropriate amount of pure water, 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (commonly known as EDC), NaCl, or KCl. By adding a material for suppressing electrostatic repulsion between fine particles to a concentration just before the polystyrene fine particles 3 aggregate so that the fine particles can be densely distributed on the substrate. It is a figure which shows typically the state which produced the fine particle suspension 4 which mixed well and suspended fine particles for fixation. A typical example of the mixing ratio of the fine particle suspension 4 is that when a commercially available 1% concentrated undiluted fine particle suspension is used, the volume ratio is 1% fine particle suspension: pure water: 10 mM EDC = 5. : 4: 6. Since the mixing ratio of the fine particle suspension is sensitive to various conditions such as room temperature, it is preferable to search for an appropriate mixing ratio by trial and error every time it is created. In order to obtain a concentration at which fine particles can be distributed at high density on the substrate, the concentration of the fine particles is preferably 0.01% or more.

  FIGS. 1C and 1D show a fine particle suspension 4 in which a fine particle suspension 4 is dropped on a gold 2 layer of a 35 mm plastic Petri dish 1 having gold 2 deposited at a thickness of 10 nm to 30 nm on the bottom surface. It is the top view and perspective view which show the state which distributed. Here, the fine particles 3 are shown in a uniformly distributed state, but it goes without saying that the fine particles 3 actually vary appropriately.

  FIG.1 (e) is sectional drawing seen in the arrow direction in the AA position of FIG.1 (c).

  A fine particle suspension 4 is dropped on a gold 2 layer of a 35 mm plastic Petri dish 1 in which gold 2 is deposited to a thickness of 10 nm to 30 nm on the bottom surface shown in FIGS. An example of a specific procedure for distributing the is as follows.

  The fine particle suspension 4 is dropped on the gold 2 layer of the 35 mm plastic Petri dish 1 and reacted at room temperature for an appropriate time, for example, 3 minutes. As long as this time is as long as 3 minutes, it is not necessary to make it longer. Thereafter, the fine particle suspension 4 is washed away with pure water, and the 35 mm plastic petri dish 1 is washed and dried well. As a result, a fine particle-fixed 35 mm plastic petri dish 1 in which the fine particles 3 are fixed on the gold 2 layer is obtained. It is assumed that one size of fine particle is fixed on one 35 mm plastic petri dish. When fixing a plurality of different fine particle diameters, a plurality of 35 mm plastic petri dishes are prepared in advance. Different fine particles should be fixed to each 35 mm plastic petri dish.

  In the present invention, there is no restriction on the size of the fine particles to be produced. For example, if fine particles having a diameter of 10 nm to 150 nm are prepared in increments of 5 nm, fine particles having different diameters in 28 steps can be prepared. Can be used as a diameter marker.

In FIG. 2A, the surface of the fine particles 3 fixed on the gold 2 layer on the bottom of the 35 mm plastic Petri dish 1 is modified with an arbitrary metal or semiconductor by a resistance heating vapor deposition apparatus. conceptual diagram illustrating a method, FIG. 2 (b) is a sectional view schematically showing an example of a specific structure of the vapor deposition source container _61.

As shown in FIG. 2A, first, resistance heating type vapor deposition is performed so that the fine particles 3 fixed on the gold 2 layer on the bottom of the 35 mm plastic Petri dish 1 fixed to the fine particles face the vapor deposition source 6. Set in the chamber of the device. The degree of vacuum in the chamber is, for example, 5 × 10 ⁻⁶ Pascal, and the temperature in the chamber is room temperature. A shutter 7 is provided between the fine particle fixed 35 mm plastic petri dish 1 and the vapor deposition source 6. The vapor deposition source 6 includes a vapor deposition source container 6 ₁ and a heating resistor 6 ₂ . The shutter 7 can be moved as indicated by the arrow displayed on the left side of the figure. When the shutter 7 covers the entire surface of the 35 mm plastic petri dish 1 on which fine particles are fixed, vapor deposition on the fine particles 3 is prevented. When the 35 mm plastic Petri dish 1 having a fine particle fixed thereon is exposed to the vapor deposition source 6 by the movement of, vapor deposition onto the fine particle 3 is performed. Metal evaporation source container ₆₁ to be deposited on the surface of the microparticles 3 or the semiconductor element is placed. Heating resistor 6 ₂ heats the element placed in the vapor deposition source container _61, a resistor to vaporize.

In FIG. 2 (b), showing an example of a specific structure of the vapor deposition source container ₆₁ schematically. In this example, the vapor deposition source container ₆₁ is a two-stage structure, the partition plate 6 ₃ is provided in the middle portion. When SiO is put into the lower part of the partition plate 6 ₃ and heated by the heating resistor 6 ₂ , the SiO evaporates and becomes SiO vapor, passing through the upper step part of the partition plate 6 ₃ , and the SiO vapor from the center into the chamber To leak. When depositing the SiO vapor to the surface of the microparticles 3 is different from the vapor of other metallic elements that travels straight particles 3 from the deposition source container _61, the process proceeds to irregularly direction of the fine particles 3 from the deposition source container 6 ₁ Therefore, during vapor deposition, SiO vapor wraps around the entire periphery of the fine particles 3. Therefore, modified fine particles 3 in which the SiO ₂ film covers almost the entire fine particles 3 can be produced.

Examples of elements that can be used as a deposition source (modifying element) 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.

  When the microparticles are polystyrene microparticles encapsulating a magnetic material, the encapsulated magnetic material gives noise to the elemental spectrum image detected from the energy dispersive characteristic X-ray detector 40-10 described later. It becomes. Therefore, when the fine particles are made of polystyrene particles containing a magnetic material, it is preferable not to use Fe, Co, or Ni as a vapor deposition source (modifying element).

  Examples of simple and easy-to-use elements include atomic number 29 (Cu), 47 (Ag), and 79 (Au).

FIGS. 3A to 3E show the surface of the fine particles 3 fixed on a single layer on the gold 2 layer on the bottom surface of the 35 mm plastic Petri dish 1 having a fine particle fixed, depending on an arbitrary metal or semiconductor. It is a conceptual diagram explaining the modification method and the modification result. Here, for clarity of illustration, showing the configuration was upside down microparticles immobilized 35mm plastic petri dish 1 and the evaporation source container ₆₁ shown in FIG.

In FIG. 3 (a), the shutter 7 disposed on the fine particles 3 _{1 to} 3 ₈ fixed on a single layer on the gold 2 layer on the bottom surface of the 35mm plastic Petri dish 1 fixed with fine particles is shown intermittently. Is moved to the left. Here, it is assumed that the distance of movement corresponds to the width of the row of two fine particles. From the upper surface of the shutter 7, as shown by an arrow 8, elemental vapor for modifying the surface of the fine particles 3 is supplied.

FIG. 3B is a diagram conceptually showing the result of the modification of the fine particles 3 _{1-3 2} with the modifying element by removing the shutter 7 at the position corresponding to the row of fine particles 3 ₁ , 3 ₂ . At this stage, since the shutter 7 is removed and only the row of fine particles 3 ₁ and 3 ₂ is exposed to the vapor of the modifying element, only the fine particles in the row of fine particles 3 ₁ and 3 ₂ are covered with the layer 9 of the modifying element. ₁ is formed. FIG. 3 (c) the shutter 7 is further moved to the left, microparticles 3 _1, 3 ₂ in addition to the column, particulate 3 _3, 3 shutter 7 at a position corresponding to a column of ₄ excluded by microparticle 3 ₁ - 3 ₄ is a diagram conceptually illustrating the results modified by the modification element. As a result, the fine particle in the row of fine particles 3 ₁ , 3 ₂ forms a modifier element layer 9 ₂ in addition to the modifier element layer 9 ₁ . The fine particle in the row of fine particles 3 ₃ and 3 ₄ is formed with a layer 9 _{2 of the} modifying element. FIG. 3 (d) is moved to the left shutter 7 In addition to the column of the particle 3 ₁ -3 _4, fine particles _35, 3 shutter 7 at a position corresponding to a column of ₆ excluded by microparticle 3 ₁ - 3 ₆ is a diagram conceptually illustrating the results modified by the modification element. As a result, fine particles in the row of fine particles 3 ₁ , 3 ₂ form a modifier element layer 9 ₃ in addition to the modifier element layers 9 ₁ , 9 ₂ . In addition to the modifying element layer 9 ₂ , the modifying element layer 9 ₃ is formed in the rows of the particulates 3 ₃ and 3 ₄ . The fine particle in the row of fine particles 3 ₅ , 3 ₆ is formed with a modifier element layer 9 ₃ . Figure 3 (e) is moved to the left shutter 7 further comprises fine particles 3 ₁ -3 ₆ in addition to the column of particulate 3 _7, 3 shutter 7 at a position corresponding to a column of ₈ excluded by microparticle 3 ₁ - 3 ₈ is a diagram conceptually illustrating the results modified by the modification element. As a result, particles 3 _1, 3 ₂ rows of fine particles in addition to the layer 9 _1, 9 _2, 9 ₃ modifications element layer 9 ₄ modifications element is formed. In the fine particles in the row of fine particles 3 ₃ and 3 ₄ , a modifier element layer 9 ₄ is formed in addition to the modifier element layers 9 ₂ and 9 ₃ . Microparticles 3 _5, 3 ₆ rows of fine particles in addition to the layer 9 ₃ modifications element layer 9 ₄ modifications element is formed. Particles 3 _7, 3 ₈ columns of the fine particles of the layer 9 ₄ modifications element is formed. In this example, modified fine particles having different thicknesses can be created step by step with one modifying element.

  Here, if the shutter 7 is continuously moved to the left side at a predetermined speed, it is possible to obtain the modified fine particles 3 having the same size of the fine particles 3 but different in only the vapor deposition film thickness of the modifying element. . Such modified fine particles 3 that are continuously different only in the vapor deposition film thickness of the modifying element are used when, for example, biofunctional molecules are adsorbed on the surface of the modified fine particles 3 and used as a label for the inspection of biological materials. It is useful for determining the optimum deposition thickness of the modifying element for the purpose. That is, the effect as a label is evaluated in advance by using the modified fine particles 3 in which only the vapor deposition film thickness of the modifying element is continuously different, and the vapor deposition film thickness of the modifying element having a large effect is actually deposited. It can be.

FIGS. 4A to 4D show a method and modification in which the surface of the fine particles 3 fixed on the gold 2 layer on the bottom of the 35 mm plastic Petri dish 1 fixed with fine particles is modified with an arbitrary metal or semiconductor. It is a conceptual diagram explaining a result. Again, as shown in FIG. 3 (a), a shutter disposed on the fine particles 3 ₁ -3 ₈ fixed on a single layer on the gold 2 layer on the bottom surface of the 35 mm plastic Petri dish 1 fixed with fine particles. 7 is moved intermittently to the left in the figure. Here, it is assumed that the substrate moves by a distance corresponding to the width of the two fine particles. From the upper surface of the shutter 7 is steam element for modifying the surface of fine particles 3 are supplied as indicated by arrow 8, wherein the exchange element of the vapor deposition source container ₆₁ each time moving the shutter 7 Thus, the vapor of the element supplied to modify the surface of the fine particles 3 is changed.

FIG. 4A is a diagram conceptually showing a result of modifying the fine particles 3 _{1-3 2} with a modifying element by removing the shutter 7 at a position corresponding to the row of fine particles 3 ₁ , 3 ₂ . At this stage, since the shutter 7 is removed and only the row of the fine particles 3 ₁ and 3 ₂ is exposed to the above-described modifier elements, only the fine particles in the row of fine particles 3 ₁ and 3 ₂ are covered with the layer 10 of the modifier element. Is formed. 4 (b) is a shutter 7 is further moved to the left, microparticles 3 _1, 3 ₂ in addition to the column, particulate 3 _3, 3 shutter 7 at a position corresponding to a column of ₄ excluded by microparticle 3 ₁ - 3 ₄ is a diagram conceptually illustrating the results modified by the modification element. As a result, in the row of fine particles 3 ₁ , 3 ₂ , the modifying element 11 is formed in addition to the modifying element layer 10. A layer 11 of modifying elements is formed in the fine particles in the row of fine particles 3 ₃ and 3 ₄ . FIG. 4 (c) are moved to the left shutter 7 further comprises fine particles 3 ₁ -3 in addition to the _fourth column, particle 3 _5, 3 shutter 7 at a position corresponding to a column of ₆ is removed by particles 3 ₁ - 3 ₆ is a diagram conceptually illustrating the results modified by the modification element. As a result, in the row of fine particles 3 ₁ , 3 ₂ , a modifier element layer 12 is formed in addition to the modifier element layers 10, 11. In addition to the modifying element layer 11, the modifying element layer 12 is formed in the rows of the fine particles 3 ₃ and 3 ₄ . A layer 12 of the modifying element is formed in the fine particles in the row of the fine particles 3 ₅ and 3 ₆ . FIG. 4 (d) is moved to the left shutter 7 further comprises fine particles 3 ₁ -3 ₆ in addition to the column of particulate 3 _7, 3 shutter 7 at a position corresponding to a column of ₈ excluded by microparticle 3 ₁ - 3 ₈ is a diagram conceptually illustrating the results modified by the modification element. As a result, the fine particle in the row of fine particles 3 ₁ , 3 ₂ forms a modifier element layer 13 in addition to the modifier element layers 10, 11, 12. In the fine particles in the row of fine particles 3 ₃ and 3 ₄ , in addition to the modifier element layers 11 and 12, a modifier element layer 13 is formed. In addition to the modifying element layer 12, the modifying element layer 13 is formed in the rows of the fine particles 3 ₅ and 3 ₆ . A layer 13 of modifier elements is formed in the fine particles in the row of fine particles 3 ₇ and 3 ₈ .

FIG. 5 shows a modification in which the surface of the fine particle 3 fixed on the gold 2 layer on the bottom of the 35 mm plastic Petri dish 1 shown in FIG. 4 (d) is modified with an arbitrary metal or semiconductor. the outermost layer of the microparticles is a conceptual diagram illustrating a result of modified with SiO ₂ layer 14. The SiO, the vapor deposition source container ₆₁ of a specific structure as shown in FIG. 2 (b) is supplied to the surface of the modified fine particles 3 of SiO vapor, of any metal or a modified modified fine particles 3 on the surface of a semiconductor The whole is covered with a layer of SiO ₂ as described above.

  FIG. 6 shows that the modified fine particles 3 fixed on a single layer on the gold 2 layer on the bottom surface of the 35 mm plastic Petri dish 1 fixed with fine particles and vapor-deposited are peeled off from the gold 2 layer on the bottom surface of the Petri dish 1. It is a conceptual diagram explaining a specific measure. For example, 200 μL of pure water is dropped from the modified fine particles 3 fixed on the gold 2 layer on the bottom surface of the Petri dish 1. The amount of pure water to be dropped may be appropriate and may be arbitrarily selected within a range of about 50 to 1000 μL. Further, a glass cell 15 having a flat bottom surface with a diameter of about 1 cm and a thickness of about 5 mm is placed on the fixed modified fine particles 3. The size of the glass cell 15 may be appropriate and may be arbitrarily selected within a range of about 3 mm to 2 cm. Next, only the bottom surface portion of the fine particle-fixed 35 mm plastic Petri dish 1 in which the glass cell 15 is placed on the modified fine particle 3 on which pure water is dropped is placed in the ultrasonic cleaning tank 16 in which water 18 is stretched. Hold on the surface.

  An ultrasonic wave is generated from the ultrasonic vibrator 17 disposed at the bottom of the ultrasonic cleaning tank 16, and a flat glass cell 15 having a flat bottom surface placed on the fixed modified fine particle 3 is indicated by an arrow. As described above, it is appropriately moved on the fixed modified fine particles 3. As a result, the modified fine particles 3 can be separated from the gold 2 layer on the bottom surface of the Petri dish 1 and suspended in pure water dropped to obtain a modified fine particle suspension. Here, the glass cell 15 does not necessarily need to be glass, and may be any material that is appropriately hard and has a flat bottom surface.

  Here, when the modified fine particle 3 is used as a label for the inspection of a biological substance, post-treatment such as adsorbing a biological functional molecule such as protein or DNA on the surface of the modified fine particle is necessary. In order to facilitate this, various processes are preferably performed as follows in each process.

  (1) The purified fine particles 3 that are fixed to the substrate are dropped into pure water, for example, as protein, bovine serum albumin (BSA), and as buffer, citrate buffer (standard sodium). citrate (SSC)) or phosphate buffer, and post-treatment of modified microparticles 3 by adding sodium dodecyl sulfate (SDS) or tannic acid as a surfactant To make it easier.

(2) For the purpose of adsorbing biological functional molecules such as protein and DNA on the surface of the modified fine particle 3, when the modifying element is deposited on the fine particle 3, the outermost surface of the deposited modifying element is SiO ₂ , Au, Ag. An element having a high affinity with biofunctional molecules such as Ni, Co, etc. Alternatively, as can be seen from FIG. 3 and FIG. 4, the modified fine particles 3 are covered with the modifying element only in the hemispherical portion on the side irradiated with the modifying element during vapor deposition. Therefore, the target surface for adsorbing biofunctional molecules such as protein and DNA on the surface of the modified fine particle on the surface of the fine particle made of polystyrene, which is the basis of the modified fine particle 3, or the fine particle made of polystyrene containing the magnetic substance. It is good to do. In this case, the polystyrene fine particles may be modified with biomolecules before the modifying element is deposited.

(3) When SiO ₂ is deposited, as can be seen from FIG. 5, the modifying element wraps around the entire fine particles 3 during the vapor deposition, so the SiO ₂ film covers almost the entire modified fine particles, so that the modification is uniformly modified. Suitable for the production of fine particles. In this case, the molecule is modified by introducing an amino group or a thiol group on the surface of the modified fine particle 3 by any of the procedures shown in (4) and (5) below.

  (4) As described above, the modified fine particle suspension in which the modified fine particles 3 are peeled off from the gold 2 layer on the bottom surface of the Petri dish 1 by using ultrasonic waves and a glass cell and suspended in the dropped pure water. An amino group is introduced to the surface of the modified fine particles by adding 0.01% to 10% or less of a silane coupling agent such as 3-aminopropyl trimethoxy silane, 3-aminopropyl triethoxy silane, and 3-aminopropyl diethoxy methyl silane. Alternatively, a thiol group is introduced on the surface of the modified fine particles by adding 0.01% to 10% or less of a silane coupling agent such as 3-mercaptopropyl trimethoxy silane.

  (5) As described in FIGS. 3 and 4, the surface of the fine particles 3 fixed on the gold 2 layer on the bottom of the 35 mm plastic Petri dish 1 fixed with fine particles is modified with any metal or semiconductor. Liquid droplets of silane coupling agents such as 3-aminoprophyl trimethoxy silane, 3-aminoprophyl triethoxy silane, and 3-aminoprophyl diethoxy methyl silane beside the modified fine particles 3 of the 35 mm plastic Petri dish 1 to which the modified fine particles 3 are immobilized Is sealed in a nitrogen or argon atmosphere container, and amino groups are introduced onto the surface of the modified fine particles 3 by adsorbing silane coupling agent molecules on the surface of the modified fine particles in the gas phase. Alternatively, a droplet of a silane coupling agent such as 3-mercaptopropyl trimethoxy silane is dropped beside the modified fine particle 3 and sealed in a nitrogen or argon atmosphere container to adsorb the silane coupling agent molecule on the surface of the fine particle in the gas phase. To introduce a thiol group onto the surface of the fine particles. In this case, it goes without saying that after introducing an amino group or a thiol group on the surface of the modified fine particle 3, the modified fine particle 3 is peeled off from the gold 2 layer as described with reference to FIG.

  In this way, after the treatment of introducing an amino group or thiol group on the surface of the modified fine particle 3, biofunctional molecules such as protein and DNA are adsorbed on the surface of the modified fine particle 3 by the methods listed below. Biofunctional molecule-modified fine particles as a label are prepared.

  (6) 0.1 to 1 g / mL of Sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] -propionamido with respect to fine particles having amino groups introduced into the surface by the treatment shown in the above (3) to (5) ) Activated using a cross-linking agent that crosslinks amino and thiol groups such as hexanoate (Sulfo-LC-SPDP) or Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC) Thereafter, DNA or RNA having a thiol group introduced at either 3 ′ or 5 ′ end, or a protein such as an antibody having a thiol group is reacted to immobilize the biofunctional molecule on the surface of the fine particles.

  (7) 0.1 to 1 g / mL of Sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] -propionamido is applied to the fine particles having a thiol group introduced on the surface by the treatment described in (3) to (5) above. ) Activated using a cross-linking agent that crosslinks amino groups with thiol groups such as hexanoate (Sulfo-LC-SPDP) or Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC) Thereafter, DNA or RNA introduced with an amino group at either 3 ′ or 5 ′ end, or a protein such as an antibody having an amino group is reacted to immobilize the biofunctional molecule on the surface of the fine particles. If necessary, the two-strand crosslinker is made to react with the fine particles after first reacting with the biofunctional molecule.

  (8) When Au or Ag is vapor-deposited on the outermost metal layer of the modified fine particle 3, DNA or RNA having a thiol group introduced at either 3 'or 5' end or a thiol group Proteins such as antibodies are directly reacted with the deposited metal on the outermost layer of the modified microparticles 3 to immobilize biofunctional molecules on the outermost layer metal deposited on the surface of the microparticles.

  (9) When Ni or Co is vapor-deposited on the outermost metal layer of the modified fine particle 3, histidine-introduced protein is directly reacted with the outermost vapor-deposited metal of the modified fine particle 3 to be deposited on the surface of the fine particle. Immobilize biofunctional molecules on the outermost metal layer.

As described above, the fine particles 3 are modified with an arbitrary transition metal, semiconductor, or metal, and the outermost layer is further covered with SiO ₂ to form modified fine particles 3. If necessary, the outermost layer of the modified fine particles 3 is modified. An amino group or a thiol group is introduced into the outer layer, and biofunctional molecules are immobilized on the outermost layer of the modified microparticles 3 to produce biofunctional molecule-modified microparticles. Thus, the produced biofunctional molecule-modified microparticles are used as a label for detecting target molecules inside and outside the cell, or target molecules extracted from cells immobilized on a substrate.

  FIG. 7 is a diagram schematically showing a configuration example of an observation target chip 19 that uses the biofunctional molecule-modified fine particle 3 of the present invention as a label for the inspection of a biological substance. A large number of probes (for example, DNA molecules) are fixed to the surface of the probe fixing region 20 of the chip 19 to be observed. Here, for simplification, each of the fixed probes 21 to 24 has a target. It is assumed that molecules (for example, target DNA, mRNA, protein) 26-29 are bound and these molecules are labeled with the modified microparticles 36-39 of the examples via the biofunctional molecules 31-34. In this example, biofunctional molecule-modified microparticles 3 suspended in a 1 mM phosphate buffer, 500 mM NaCl, and 0.5% SDS solution are dropped onto the probe fixing region 20 of the chip 19 to be observed, and are dropped at 40 ° C. for 16 hours. React. Then, it is washed with the same solvent. As a result, for example, the biofunctional molecule-modified microparticles 3 modified with a biofunctional molecule that specifically hybridizes with the target DNA are immobilized on the target DNA and function as a label. The sample subjected to the reaction is sufficiently dried and observed. Here, for simplification, an example of capturing a nucleic acid molecule such as DNA as a target molecule has been taken. However, the probe molecule immobilized on the surface of the probe immobilization region 20 is not limited to DNA, and has an affinity for the target molecule. In consideration of the above, RNA and protein molecules are appropriately selected and fixed. Moreover, the assumption as a target molecule captured by the target capturing molecules 21-24 immobilized on the probe fixing region 20 is all molecules that control cell functions such as DNA, RNA, and protein. The solvent composition, temperature, and reaction time during the reaction must be determined each time so that these target molecules react most efficiently with the capture molecules 21-24 and the particulate biofunctional molecules 31-34 on the substrate. . In particular, when a protein is assumed and detected as a target molecule, special consideration must be given to the solvent composition and temperature so that the three-dimensional structure and activity are preserved.

For example, the modified fine particles 30 ₁ , 30 ₂ , 30 ₃ , 30 ₄ , 30 ₅ ,... For comparison are arranged at positions separated from the surface of the probe fixing region 20 of the tip 19 to be observed, for example, at the periphery of the tip 19. -Is provided. It goes without saying that these modified fine particles 30 ₁ , 30 ₂ , 30 ₃ , 30 ₄ , 30 ₅ , ---- are known to the observer what the modifying elements are.

  In order to perform the electron microscope observation on the observation target chip 19, it is processed according to a normal electron microscope sample preparation procedure, and then inserted into the sample region of the electron microscope for observation. If the sample is not electrically conductive, pre-treatment is performed by pre-depositing carbon, tungsten, gold, platinum, etc. on the surface about 1-5 nm so that the entire sample is covered with carbon, tungsten, gold, platinum, etc. Use as a microscope sample.

  FIG. 8 illustrates an example of observing the observation target chip 19 of the embodiment described in FIG. 7 with a scanning electron microscope as a specific example of using the biofunctional molecule-modified fine particle 3 of the embodiment as a label for inspection of a biological substance. FIG.

A large number of probes (for example, DNA molecules) are fixed to the surface of the probe fixing region 20 of the chip 19 to be observed, but in FIG. 7, each of the fixed probes 21 to 24 is simplified. It is assumed that the target molecules 26-29 are bound and these molecules are labeled with the modified fine particles 36-39 of the examples via the biofunctional molecules 31-34. In this example, modified fine particles 36-39 is one layer, each modified metal with the same diameter, it is assumed that the outermost layer is covered with SiO _2. The modified fine particle 36 is modified with atomic number 29 (Cu), the modified fine particle 37 is modified with atomic number 47 (Ag), the modified fine particle 38 is modified with atomic number 79 (Au), and the modified fine particle 39 is modified with atomic number 28 (Ni). It shall be. Comparative modified fine particles 30 ₁ , 30 ₂ , 30 ₃ , and 30 ₄ are provided on the periphery of the chip 19 in a region that is scanned simultaneously with the sample to be observed. In this case, the modified fine particles 30 ₁ , 30 ₂ , 30 ₃ , and 30 ₄ for comparison are also used as labels, each modified with the same metal as the modified fine particles 36 to 39 to be detected.

  On the surface of the probe fixing region 20 of the chip 19, an electron gun 40-1, a focusing lens 40-2, and a scanning coil 40-3 of a scanning electron microscope 40 for detecting modified fine particles are provided. Electrons of the electron beam 40-4 emitted from the electron gun 40-1 collide with the modified fine particles 36, 37, 38 and 39, and the modified fine particles emit secondary electrons 40-5 and reflected electrons 40-6. The detector 40-7 captures the secondary electrons and the detector 40-8 captures the reflected electrons. Based on the secondary electrons detected by the detector 40-7, a secondary electron beam image is obtained, and the position and size of the modified fine particles are specified. The element that modifies the position and size of the modified fine particles and the surface is specified by the reflected electron beam image obtained based on the reflected electrons detected by the detector 40-8.

  On the other hand, when the electrons of the electron beam 40-4 collide with the modified fine particles 36, 37, 38 and 39, the modified fine particles 36, 37, 38 and 39 emit X-rays 40 having a wavelength specific to the constituent elements of the modified fine particles. An energy dispersive X-ray detector 40-10 for detecting -9 is provided. That is, an element spectrum image is obtained from the wavelength signal corresponding to the constituent element for modification detected by the energy dispersive characteristic X-ray detector 40-10.

  Therefore, in the present invention, the position and size of the modified fine particles are measured by the reflected electron beam image obtained by detecting the biological functional molecule modified fine particles bonded to the probe (for example, DNA molecule) on the surface of the probe fixing region 20 of the chip 19 based on the reflected electron detection. Based on the identification of the element that modifies the surface and the surface, the position and size information of the modified fine particles based on the secondary electron beam image is used as necessary. Furthermore, if necessary, the element type is used to identify the type of element deposited on the surface of the modified fine particle. The type of microparticle, that is, the type of target molecule on the inside or outside of the cell or on the substrate, which binds to the biofunctional molecule modified on the surface of the microparticle is specified.

  9A to 9D show the surface of the modified fine particle from the reflected electron beam image, the secondary electron beam image, the element spectrum image, and the element mapping image on the surface of the probe fixing region 20 of the tip 19 to be observed. It is a figure which shows typically the specific example which identifies the kind of element currently vapor-deposited.

FIG. 9A is a diagram schematically showing a reflected electron beam image of the modified fine particles to be detected as a label on the surface of the probe fixing region 20 of the tip 19 to be observed. Here, the modified fine particles shown in FIG. It shows that an image of reflected electrons corresponding to the modifying element 36-39 and an image of reflected electrons corresponding to the modifying element of the modified fine particles 30 _{1 to} 30 ₄ for comparison are obtained. The number of reflected electrons increases as the modifying element of the modified fine particles becomes heavier, and the reflected electron image decreases as it becomes lighter. That is, the heavier the modifier, the brighter it looks. Therefore, if the brightness of the reflected electrons corresponding to the modifying element of the modified fine particles according to the setting of the scanning electron microscope 40 is evaluated in advance, the modified fine particles can be identified by the properties by the reflected electron beam image.

On the other hand, depending on the mode of use of the scanning electron microscope 40, the brightness seen by the observer may vary even with the same modifying element, and when the number of modifying fine particles used increases, It is considered that it is difficult to identify the modified fine particles simply by looking at the brightness. For this reason, modified fine particles 30 ₁ , 30 ₂ , 30 ₃ , 30 ₄ for comparison are provided in a region where electron beam scanning is performed simultaneously with the sample to be observed. In this case, the modified fine particles 30 ₁ , 30 ₂ , 30 ₃ , and 30 ₄ for comparison are also used as labels, each modified with the same metal as the modified fine particles 36 to 39 to be detected. As a result, the brightness of the reflected electron beam image of the sample to be observed can be evaluated based on the brightness of the modified fine particles for comparison. That is, the modification element of the modified fine particle can be identified based on the brightness of the modified fine particle to be detected as a label and the brightness of the modified modified fine particle as a reference. The reflected electron beam image of the modified fine particles for comparison is extremely useful when the number of modified fine particles used as a label increases and there is a small contrast of the reflected electron beam image of the modified fine particles.

As a matter of course, the modified fine particles 30 ₁ , 30 ₂ , 30 ₃ , 30 ₄ , --- for comparison indicate in which position the modified fine particles having which modifying element are arranged in the manufacturing process of the chip 19. It is something that will be made clear.

  FIG. 9B is a diagram schematically showing a secondary electron beam image of the modified fine particles to be detected as a label on the surface of the probe fixing region 20 of the tip 19 to be observed. The secondary electron beam image can be obtained as a clear image regardless of the modifying element of the modified fine particles. On the other hand, since the reflected electron beam image has a small contrast and is displayed as a dark image, it is difficult to determine whether the fine particles modified with a light element have modified fine particles or the size is difficult to determine. That happens. In such a case, by referring to the secondary electron beam image at that position, it is possible to clearly determine that there is a modified fine particle at that position or the size of the modified fine particle.

  FIG. 9C is a diagram schematically showing an element spectrum image of the modified fine particles to be detected as a label on the surface of the probe fixing region 20 of the tip 19 to be observed. The horizontal axis represents wavelength, and the vertical axis represents signal intensity. In this example, it can be seen that there are four types of modified fine particle elements used as labels.

  FIG. 9D is a diagram schematically showing a mapping image of elements of the modified fine particles to be detected as a label on the surface of the probe fixing region 20 of the tip 19 to be observed. The mapping image of this element is obtained by mapping the signal regarding the modifying element obtained in the energy dispersive X-ray detector 40-10 in the same manner as the secondary electron beam image shown in FIG. 9B. Similar to the image, not only the size and position of the modified fine particles can be shown, but the image can be used as an image having information on the modifying element. However, compared with a secondary electron beam image, it takes a relatively long time to collect data as well as a relatively low spatial resolution.

  9A to 9D, only four modified fine particles have been described. However, in the present invention, for example, if fine particles having a diameter of 10 nm to 150 nm are prepared in increments of 5 nm, 28 Fine particles with different step diameters can be created. In addition, these fine particles can be used as labels having different particle sizes with respect to the same material for the above-described modification with the metal or semiconductor, so that very many kinds of labels can be produced.

FIGS. 10A to 10D show the case where the elements deposited on the surface of the modified fine particles are Au and Ag, and the outermost layer thereof is covered with SiO _2. It is a figure which shows typically the example of the aspect of reflection of this. Here, the atoms of the constituent elements of each layer are indicated by small circles. 10A and 10B, a modified metal layer is formed in the order of Ag and Au from the surface of the fine particles, and the outermost layer is covered with SiO ₂ . In FIGS. 10C and 10D, the modified metal layers are formed in the order of Au and Ag from the surface of the fine particles. The outermost layer is covered by the SiO _2.

  Comparing FIG. 10A and FIG. 10B, the difference between whether the electrons of the electron beam 40-4 emitted from the electron gun 40-1 are reflected by the Au layer or the Ag layer is different. There is. Therefore, even if the same labeling fine particles are used, they differ from the reflection on the Au layer or the reflection on the Ag layer due to the difference in energy of the electron beam 40-4, and the brightness seen by the observer is different. become. Similarly, when FIG. 10C and FIG. 10D are compared, electrons of the electron beam 40-4 emitted from the electron gun 40-1 are reflected by the Ag layer or reflected by the Au layer. There is a difference. Therefore, even with the same modified fine particles for labeling, the reflection on the Ag layer or the reflection on the Au layer differs from the reflection on the Au layer due to the energy difference of the electron beam 40-4, and the brightness seen by the observer is different. It will be.

  As described above, in order to prevent the observation result from being different due to the difference in energy of the electron beam 40-4 irradiated to the labeling modified fine particles, before observation, for example, the brightness of the known modified fine particles for comparison is increased. It is preferable to adjust the energy of the electrons of the electron beam 40-4 emitted from the electron gun 40-1. Alternatively, prior calibration may be omitted, and observation may be performed in comparison with the brightness of known modified fine particles for comparison.

  In the example shown in FIGS. 10A to 10D, the modification order is different between FIGS. 10A and 10B and FIG. 10C and FIG. Since the elements are only Au and Ag, the element spectral images of the modified fine particles described with reference to FIG. 9 are the same, but the element mapping image reflects the difference between Au and Ag.

As described above, the present invention can provide fine particles for labeling in which fine particles having an arbitrary particle diameter are modified with a thin film of any transition metal, metal, or semiconductor and the outermost layer is wrapped with SiO ₂ . Therefore, not only can various types of labels be easily created, but the outermost layer of fine particles is wrapped with SiO ₂ , so that the outermost layer of fine particles modified with any transition metal, semiconductor, or metal can have amino groups, or The thiol group can be introduced by a unified method. As a result, biofunctional molecule-modified fine particles can be produced by stably immobilizing biofunctional molecules on the outermost layer of the modified fine particles.

(A)-(e) is a conceptual diagram explaining the process which fixes fine particle on the board | substrate of the first process of the modified fine particle preparation process of this invention. (A) is a method in which the surface of the fine particles 3 fixed on the gold 2 layer on the bottom of the 35 mm plastic petri dish 1 is modified with an arbitrary metal or semiconductor by a resistance heating type vapor deposition apparatus. description conceptual diagram, (b) is a sectional view schematically showing an example of a specific structure of the vapor deposition source container _61. (A)-(e) modify the surface of the fine particles 3 fixed on the gold 2 layer on the bottom of the 35 mm plastic Petri dish 1 with a different thickness by an arbitrary metal or semiconductor. It is a conceptual diagram explaining a method and a modification result. (A)-(d) shows a method of modifying the surface of the fine particle 3 fixed on the gold 2 layer on the bottom of the 35 mm plastic Petri dish 1 with a fine particle fixed with an arbitrary metal or semiconductor and the modification result. It is a conceptual diagram to explain. The outermost layer of modified fine particles obtained by modifying the surface of the fine particles 3 fixed on the gold 2 layer on the bottom surface of the 35 mm plastic Petri dish 1 shown in FIG. 4D with an arbitrary metal or semiconductor. the is a conceptual diagram illustrating a result of modified with SiO ₂ layer 14. Describes a specific measure for peeling the modified fine particles 3 fixed on the bottom of the gold 2 layer on the bottom of the Petri dish 1 from the gold 2 layer on the bottom of the Petri dish 1. FIG. It is a figure which shows typically the structural example of the chip | tip 19 of an example of an observation object which utilizes the biofunctional molecule modification fine particle 3 of this invention as a label | marker for the test | inspection of a biological substance. 7 is a conceptual diagram illustrating an example of observing the observation target chip 19 of the mode described in FIG. 7 with a scanning electron microscope as a specific example in which the biofunctional molecule-modified fine particle 3 of the embodiment is used as a label for inspection of a biological substance. is there. (A)-(c) shows the types of elements deposited on the surface of the modified fine particles from the reflected electron beam image, secondary electron beam image, and element spectrum image of the surface of the probe fixing region 20 of the tip 19 to be observed. It is a figure which shows typically the specific example to identify. (A)-(d) shows the case where the elements deposited on the surface of the modified fine particles are Au and Ag, and the outermost layer thereof is covered with SiO _2. It is a figure which shows the example of this aspect typically.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... 35mm plastic petri dish, 2 ... Gold thin film, 3 ... Polystyrene fine particle, 4 ... Fine particle suspension, 5 ... Container with lid, 6 ... Deposition source, 6 ₁ ... Deposition source container, 6 ₂ ... For heating Resistor, 6 ₃ ... partition plate, 7 ... shutter, 9 ₁ , 9 ₂ , 9 ₃ , 9 ₄ ... layer of modifying element, 10, 11, 11, 12, 13, 14 ... layer of modifying element, 15 ... glass Cell: 16 ... ultrasonic cleaning tank, 17 ... ultrasonic transducer, 18 ... water, 19 ... chip to be observed, 20 ... probe fixing region, 21-24 ... fixed probe, 26-29 ... target molecule, 30 ₁ , 30 ₂ , 30 ₃ , 30 ₄ , 30 ₅ ... modified fine particles, 31-34 ... biofunctional molecules, 36-39 ... modified fine particles, 40-1 ... electron gun, 40-2 ... focusing lens, 40-3 ... Scanning coil, 40-4 ... Electron beam, 40-5 ... Secondary electron, 40-6 ... Backscattered electrons, 40-7 ... Secondary electron detector, 40-8 ... Backscattered electron detector, 40-9 ... X-rays with specific wavelength for X-ray elements with specific wavelength for constituent elements of modified fine particles 40-10 Energy dispersive characteristic X-ray detector.

Claims (18)

Preparing a substrate, a silicon substrate, or a glass substrate having a gold thin film formed on one surface;
Suspension of fine particles by mixing any one of polystyrene fine particles having a predetermined diameter, SiO ₂ fine particles, or TiO ₂ fine particles, an appropriate amount of pure water, and a material for suppressing electrostatic repulsion between the fine particles. Preparing the liquid,
Dropping the fine particle suspension onto the substrate to distribute the fine particles on the substrate;
Cleaning and removing excessive amounts of fine particles not adsorbed on the substrate, and drying the fine particles distributed on the substrate;
Depositing at least one layer of transition metal, metal or semiconductor on the fine particles distributed on the substrate;
A step of forming one layer of SiO ₂ by vapor deposition or sublimation on the fine particles on which at least one layer of the transition metal, metal or semiconductor is vapor-deposited;
On the fine particles fixed on the substrate and the vapor-deposited,
(I) Trace amount of pure water, or (ii) Trace amount of pure water, bovine serum albumin (BSA) as protein, and citrate buffer (standard sodium citrate (SSC)) or phosphate buffer as buffer (Phosphate buffer), a step of dripping a small amount of liquid containing sodium dodecyl sulfate (SDS) or tannic acid as a surfactant, and ultrasonic action on the other surface of the substrate Then, a material having a flat bottom surface on the one surface side to which the fine particles are fixed is placed so that a slight load is applied to the bottom surface, and the fine particles are moved to the substrate by moving the material in an arbitrary direction. Peeling from
Of fine particles modified with transition metals, metals or semiconductors.   2. The method for producing fine particles according to claim 1, wherein the material for suppressing electrostatic repulsion between the fine particles is 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride, NaCl, or KCl.   The transition metal, metal, or semiconductor is a transition metal up to 79, excluding atomic number 43 in the periodic table, atomic number 13, 31, 32, 33, 49, 50, 51, 81, 82, or 83. 2. The method for producing fine particles according to claim 1, wherein the fine particles are any one of a metal and a semiconductor having an atomic number of 14, 34, or 52.   Silane coupling of 0.01% to 10% of 3-aminopropyl trimethoxy silane, 3-aminopropyl triethoxy silane, or 3-aminopropyl diethoxy methyl silane to the fine particle suspension obtained by the step of peeling the fine particles from the substrate. The method for producing fine particles according to claim 1, wherein an amino group is introduced into the surface of the fine particles by adding an agent.   A thiol group is introduced to the surface of the fine particles by adding 0.01% to 10% or less of a silane coupling agent of 3-mercaptopropyl trimethoxy silane to the fine particle suspension obtained by the step of peeling the fine particles from the substrate. The method for producing fine particles according to claim 1. Preparing a substrate with a gold thin film formed on one side, a silicon substrate, or a glass substrate;
Suspension of fine particles by mixing any one of polystyrene fine particles having a predetermined diameter, SiO ₂ fine particles, or TiO ₂ fine particles, an appropriate amount of pure water, and a material for suppressing electrostatic repulsion between the fine particles. Preparing the liquid,
Dropping the fine particle suspension onto the substrate to distribute the fine particles on the substrate;
Depositing at least one layer of transition metal, metal or semiconductor on the fine particles distributed on the substrate;
(I) 3-aminoprophyl trimethoxy silane, 3-aminoprophyl triethoxy silane, or 3-aminoprophyl diethoxy methyl silane silane coupling around the substrate having the fine particles deposited with at least one layer of the transition metal, metal or semiconductor A droplet of the agent is dropped and sealed in a nitrogen or argon atmosphere container, and an amino group is introduced to the surface of the fine particle by adsorbing the silane coupling agent molecule on the fine particle surface in the gas phase, or ( ii) Dropping 3-mercaptopropyl trimethoxy silane silane coupling agent droplets, sealing in a nitrogen or argon atmosphere container, and adsorbing the silane coupling agent molecules on the fine particle surface in the gas phase A step of introducing a thiol group into
After dropping a small amount of pure water onto the fine particles fixed and deposited on the substrate, ultrasonic waves are applied to the other surface of the substrate, and the fine particles on the single layer are fixed. A step of separating the fine particles from the substrate by placing a material having a flat bottom surface on the surface so as to apply a load to the bottom surface and moving the material in an arbitrary direction;
Of fine particles modified with transition metals, metals or semiconductors.   The fine particle suspension is suspended so as to have a concentration immediately before the fine particles aggregate so that the fine particles can be distributed at a high density on the substrate. How to make fine particles. After introducing amino groups on the surface of the fine particles , 0 . 1 mg to 1 g / mL Sulfosuccinimidyl 6- (3 '-[2-pyridyldithio] -propionamido) hexanoate (Sulfo-LC-SPDP) or Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC) After activation with a cross-linking agent that crosslinks the amino group and thiol group, DNA or RNA introduced with a thiol group at either the 3 'or 5' end, or an antibody protein having a thiol group reacts The method for producing fine particles according to claim 4, wherein a biofunctional molecule is immobilized on the surface of the fine particles. After introducing the thiol group to the surface of the fine particles , 0 . 1 mg to 1 g / mL Sulfosuccinimidyl 6- (3 '-[2-pyridyldithio] -propionamido) hexanoate (Sulfo-LC-SPDP) or Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC) After activation using a cross-linking agent that crosslinks the thiol group and amino group of DNA, DNA or RNA with amino group introduced at either 3 'or 5' end, or antibody protein having amino group reacts 6. The method for producing fine particles according to claim 5, wherein the biofunctional molecule is immobilized on the surface of the fine particles.   The method for producing fine particles according to claim 8 or 9, wherein the two-crosslinking agent is reacted with the fine particles after first reacting with a biofunctional molecule.   After introducing amino groups on the surface of the fine particles, before removing the fine particles from the substrate, 0.1 mg to 1 g / mL of Sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] -propionamido) hexanoate (Sulfo-LC -SPDP), or after activation with a cross-linking agent that crosslinks the amino group and thiol group of Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC), 3 'or 5 7. The method for producing microparticles according to claim 6, wherein a biofunctional molecule is immobilized on the surface of the microparticle by reacting DNA or RNA having a thiol group introduced at either end thereof or a protein of an antibody having a thiol group.   After introducing a thiol group on the surface of the fine particles and before peeling the fine particles from the substrate, 0.1 mg to 1 g / mL of Sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] -propionamido) hexanoate (Sulfo-LC -SPDP), or activated with a two-crosslinker that crosslinks the thiol group and amino group of Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC), then 3 'or 5 7. The method for producing microparticles according to claim 6, wherein the biofunctional molecule is immobilized on the surface of the microparticle by reacting DNA or RNA having an amino group introduced at either end thereof or an antibody protein having an amino group.   The method for producing fine particles according to claim 11 or 12, wherein the two-crosslinking agent is reacted with the fine particles after first reacting with a biofunctional molecule. The microparticles manufactured by the microparticle manufacturing method according to claim 1 or 6 are used, and many probes fixed to the probe fixing region on the surface of the chip to be observed contain DNA, mRNA, or protein. A comparison in which the target molecule binds and the target molecule is labeled via a biofunctional molecule on the surface of the microparticle and the surface of the probe fixing region of the observation target chip is separated. Obtaining a reflected electron beam image by electron beam scanning of the fine particles obtained by simultaneously scanning the fine particles with an electron beam,
A living body characterized in that a target molecule containing the DNA, mRNA, or protein is specified by determining a material that modifies the surface of the fine particle from the brightness of the reflected electron beam image of the labeled fine particle. Inspection method for substances. The microparticles manufactured by the microparticle manufacturing method according to claim 1 or 6 are used, and many probes fixed to the probe fixing region on the surface of the chip to be observed contain DNA, mRNA, or protein. A comparison in which the target molecule binds and the target molecule is labeled via a biofunctional molecule on the surface of the microparticle and the surface of the probe fixing region of the observation target chip is separated. Obtaining a reflected electron beam image by electron beam scanning of the fine particles obtained by simultaneously scanning the fine particles with an electron beam,
A material that modifies the surface of the fine particle is determined by comparing the reflected electron beam image of the comparative fine particle with the reflected electron beam image of the labeled fine particle, and includes the DNA, mRNA, or protein. A method for inspecting biological material, characterized by identifying a target molecule.   In addition to the reflected electron beam image obtained by scanning the electron beam with the electron beam, a secondary electron beam image is obtained from the secondary electron obtained by scanning with the electron beam. The biological material inspection method according to claim 14 or 15, wherein a position of the fine particle is specified.   In addition to the reflected electron beam image of the fine particles obtained by scanning with the electron beam, the secondary electron beam image obtained from the secondary electrons obtained by scanning with the electron beam, The biological material inspection method according to claim 16, wherein an element spectrum image of a material that modifies the surface of the obtained fine particles is obtained, and a composition element of the fine particles is specified.   In addition to the reflected electron beam image obtained by scanning the electron beam with the electron beam and the secondary electron beam image obtained from the secondary electron obtained by scanning with the electron beam, the surface of the fine particle is modified. The biological material inspection method according to claim 16, wherein an element mapping image of a material being obtained is obtained to identify a composition element of the fine particles.

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