JP6496974B2 - Separation method of separation object and substrate for separation object separation - Google Patents

Description

  The present invention relates to a method for separating an object to be separated and a substrate used for separating the object to be separated, and in particular, a method for separating an object to be separated in the form of fine particles and a substrate used for separating the object to be separated. About.

  In recent years, a microchip used for separation and analysis of microparticles has been developed by applying nanoimprint lithography (NIL) technology, which is a microfabrication technology in the semiconductor field. This microchip is equipped with a fine separation channel formed by the nanoimprint lithography technique as the microfabrication technique, and the microchip contained in the sample is introduced by introducing a very small amount of the sample into the microchip. Particle separation and analysis are possible. In particular, development of microchips used for the purpose of separating and analyzing cells, living tissues, and the like has been actively conducted.

  As such a microchip, conventionally, fine particles (for example, cells, microorganisms (bacteria, viruses, etc.), liposomes, which have a curved channel formed on a substrate and flow through the curved channel, Chromosomes, mitochondria, organelles and other biologically related microparticles; latex particles, gel particles, industrial particles, etc., synthetic particles), etc., depending on the specific gravity of the microparticles using the action of centrifugal force One that can be separated in the direction of centrifugal force has been proposed (see Patent Document 1).

JP 2009-162643 A

  However, according to the separation method using the action of centrifugal force as described above, although separation according to specific gravity is possible, it is difficult to separate each separation object (separated object) according to its size. It is. In particular, membrane vesicles (exosomes), organelles (organelles), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), granules (melanosomes, microsomes, glyoxy) secreted from living cells or cultured cells Some biologically related microparticles such as a some, a bibel-parade body, etc.), a cytoskeleton, a central body, flagella, cilia, ribosome, virus, etc. have different specific sizes but similar specific gravity. Therefore, in the separation method using the action of centrifugal force, there is a problem that it is difficult to separate them. Among the above bio-related microparticles, membrane vesicles (exosomes) secreted from in vivo cells and present in body fluids play an important role in intracellular signal transduction, and are used for treatment of human diseases and as biomarkers. It is known to have potential. By separating the membrane vesicles (exosomes) one by one, the microRNAs possessed by each membrane vesicle (exosome) can be analyzed, and the microRNAs can be used for treatment and diagnosis. .

  Among microparticulates, biologically related microparticles such as membrane vesicles (exosomes) play an important role in intracellular signal transduction, and can be used as treatments for human diseases and as biomarkers. Therefore, there is a demand for a method for selectively separating biologically relevant microparticles such as membrane vesicles (exosomes) according to their dimensions (size).

  In view of such problems, the present invention provides a method for selectively separating a separated object according to its size (size) from a separated object mixture including a fine particle-shaped separated object, and the separation method. It is an object to provide a substrate for separating an object to be used.

In order to solve the above-mentioned problems, the present invention is a method for separating an object to be separated from a mixture of objects to be separated containing a fine particle-like object to be separated, and a recess and a protrusion having a size of 10 to 500 nm. Including the step of bringing the separated mixture into contact with the concavo-convex pattern by bringing the separated mixture into contact with a concavo-convex structure having a concavo-convex pattern, and separating the separated body from the separated mixture. the uneven pattern, the have a separation target material substantially the same size, the object separator is a membrane vesicles secreted from in vivo cell or a cultured cell, the uneven structure, quartz glass a method of separating the separator characterized that you have been composed of (invention 1).

A fine particle-like object to be separated has a property of being easily attached selectively to an uneven pattern having substantially the same dimensions as the object to be separated when brought into contact with an uneven structure having an uneven pattern. Therefore, according to the said invention (invention 1), by making a to-be-separated body mixture contact the concavo-convex structure which has an uneven | corrugated pattern of the substantially same dimension as a to-be-separated object, it is substantially the same dimension as the said uneven | corrugated pattern. An object to be separated can be selectively separated. In addition, since the size of the object to be separated is approximately 10 to 500 nm, the object to be separated easily adheres selectively by the uneven pattern having substantially the same size. It is possible to selectively separate objects to be separated having substantially the same dimensions as the pattern.

  In the present invention, “substantially the same dimension” means that the ratio of the dimension of the object to be separated and the dimension of the concavo-convex pattern is in the range of 1: 0.5 to 2.0, preferably The ratio is 1: 0.7 to 1.5, particularly preferably 1: 0.8 to 1.2.

In the said invention (invention 1 ), the area | region which contacts the said to-be-separated substance mixture is divided into several area | regions among the said uneven structure body, The dimension of the said uneven | corrugated pattern provided in each of these several area | regions is Are preferably different from each other (Invention 2 ).

In the said invention (invention 1 and 2 ), it is preferable that the surface of the said uneven structure body which contacts the said to-be-separated object mixture is a hydrophilic surface (invention 3 ). According to this invention (invention 3 ), the surface of the concavo-convex structure that comes into contact with the mixture to be separated is a hydrophilic surface, so that the separated object that easily adheres to the hydrophilic surface is selectively selected according to the size. Can be separated.

In addition, the present invention is a substrate for separating an object to be separated that is used for separating the object to be separated from a mixture to be separated containing a fine particle-like object to be separated, and the base material and one of the base materials And a concave / convex pattern having a dimension of 10 to 500 nm formed on the surface of the substrate, wherein the concave / convex pattern has substantially the same dimension as the object to be separated. Are membrane vesicles secreted from in vivo cells or cultured cells, the base material is made of quartz glass, and by contacting the separated material mixture with the separated material separation substrate, Provided is a substrate for separating an object to be separated, wherein the object to be separated is attached to the concavo-convex pattern and the separated object can be separated from the mixture of separated objects (Invention 4 ).

In the above invention (invention 4), before Symbol uneven pattern is formed in each of a plurality of regions formed by dividing the one surface of the substrate, the irregularities are provided on each of the plurality of regions the dimensions of the recess and the convex portions of the pattern, different from the preferred one another (invention 5), the surface of the concavo-convex pattern is not the preferred of a hydrophilic surface (invention 6).

  ADVANTAGE OF THE INVENTION According to this invention, the method of selectively isolate | separating a to-be-separated object from the to-be-separated substance mixture containing a to-be-separated object of a microparticulate form according to the dimension (size), and the to-be-separated object separation used for the said separation method A substrate can be provided.

FIG. 1 is a cut end view showing a schematic configuration of an object separation substrate according to an embodiment of the present invention. FIG. 2 is a plan view schematically showing another configuration of the separation target substrate according to the embodiment of the present invention. FIG. 3 is a process flow diagram showing a manufacturing process of the substrate for separating an object according to one embodiment of the present invention in a cut end view.

Embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a cut end view illustrating a schematic configuration of a separation target separating substrate according to the present embodiment, and FIG. 2 schematically illustrates another configuration of a separation target separating substrate according to the present embodiment. It is a top view.

  As shown in FIG. 1, the substrate separating substrate 1 according to this embodiment is a concavo-convex structure having a base material portion 2 and a concavo-convex pattern 3 formed on a main surface 21 of the base material portion 2. Yes, it is used to separate microparticles (separated bodies) having specific dimensions from a mixture of separated bodies containing microparticles.

  The separation target substrate 1 according to the present embodiment has a specific size of the target when the size of the fine particles contained in the separation target mixture is about 10 to 500 nm, preferably about 50 to 200 nm. It is useful for selectively separating the separated body. In particular, membrane vesicles (exosomes), organelles, vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.) secreted from in vivo or cultured cells in the form of fine particles (dimensions of about 10 to 500 nm) ), Granules (melanosomes, microbodies, glyoxisomes, vibel parade bodies, etc.), cytoskeletons, central bodies, flagella, cilia, ribosomes, viruses, etc. It is useful for selectively separating them from culture fluids such as body cells, serum, etc. according to their dimensions.

  The material constituting the substrate 2 includes quartz glass, synthetic quartz glass, soda glass, fluorite, calcium fluoride, magnesium fluoride, acrylic glass, borosilicate glass, and the like; polycarbonate, polypropylene, polyethylene, polystyrene In addition, transparent materials such as resin materials such as polyolefin can be used. In particular, it is preferable to use the transparent material as the material constituting the base material portion 2 because the separation object can be separated while observing the adhesion state of the separation object to the uneven pattern 3 with a microscope or the like. Furthermore, when a material having a hydrophilic surface such as quartz glass is used as a material constituting the base material portion 2, a separation target (for example, a membrane secreted from in vivo cells or cultured cells) that easily adheres to the hydrophilic surface. Vesicles (exosomes), organelles (organelles), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), granules (melanosomes, microscopic bodies, glyoxysomes, vibel parade bodies, etc.), cytoskeleton, A centriole, flagella, cilia, ribosome, virus, etc.) can be selectively separated.

  In the present embodiment, “transparent” means that when light having a wavelength of 190 to 800 nm is irradiated from one side of an object (in the present embodiment, the base material portion 2), the light is emitted to the side opposite to the irradiated side. It means to reach. If a suitable standard is expressed by transmittance, it is 20% or more, preferably 50% or more, particularly preferably 80% or more.

  The shape of the base material part 2 is not particularly limited, and examples thereof include a substantially rectangular shape in plan view and a substantially circular shape in plan view. Moreover, the magnitude | size of the base material part 2 in planar view and the thickness of the base material part 2 are not specifically limited, According to the use of the to-be-separated object isolation | separation board | substrate 1 etc., it can set suitably. For example, membrane vesicles (exosomes), organelles (organosomes), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), granules (melanosomes, Microspheres, glyoxisomes, bibel-parade bodies, etc.), cell skeleton, central bodies, flagella, cilia, ribosomes, cell tissues containing biological tissues such as viruses, etc. When the separated object separation substrate 1 is brought into contact with a cell culture solution contained in a container such as a petri dish to separate the separated object, the size of the separated object separation substrate 1 in a plan view is determined in the container. The size can be set so that the separation target substrate 1 can be placed. Similarly, the thickness of the separation target substrate 1 can also be set to a thickness that allows the uneven pattern 3 to come into contact with the cell culture solution when the separation target substrate 1 is placed in the container. .

The concavo-convex pattern 3 is formed on the main surface 21 of the base material part 2 as a structure integrated with the base material part 2.
The shape of the concavo-convex pattern 3 is not particularly limited, and examples thereof include a substantially circular or substantially rectangular pillar shape, a hole shape, and a line and space shape in a plan view.

  The dimension of the concavo-convex pattern 3 is substantially the same as the dimension of the separated object that is the separation object among the separated objects included in the separated object mixture. For example, when the dimension of the separation target as the separation target is about 100 nm, the dimension of the uneven pattern 3 can be set to about 50 to 200 nm, preferably about 70 to 150 nm, and particularly preferably about 80 to 120 nm. .

  The dimension of the concave / convex pattern 3 is, for example, when the concave / convex pattern 3 is a pillar-shaped convex portion 32 or a hole-shaped concave portion 31 having a substantially circular or substantially rectangular shape in plan view, and the dimension of the pillar-shaped convex portion 32 or the hole-shaped concave portion 31. The diameter or short side length is meant, and when the concavo-convex pattern 3 has a line-and-space shape, it means the width in the short direction of the line-like convex portion 32 and the space-like concave portion 31.

  In the example shown in FIG. 1, the concave portion 31 and the convex portion 32 of the concave / convex pattern 3 are provided with substantially the same dimensions, but the present invention is not limited to such an embodiment, and the concave portion 31 and the convex portion 32 of the concave / convex pattern 3. The dimensions may be different. For example, the dimension of the convex part 32 of the concavo-convex pattern 3 may be 100 nm, and the dimension of the concave part 31 may be 300 nm. By doing in this way, the to-be-separated body with a dimension of about 100 nm can be attached to the convex part 32 of the uneven | corrugated pattern 3, and the to-be-separated body with a dimension of about 300 nm can be made to adhere to the recessed part 31, They can be selectively separated by alternating arrangement.

  The height (aspect ratio) of the concavo-convex pattern 3 is not particularly limited and can be appropriately set according to the dimensions of the concavo-convex pattern 3. Usually, the height of the concavo-convex pattern 3 can be set so that the aspect ratio of the concavo-convex pattern 3 is about 0.5 to 5.

  The main surface 21 of the base material portion 2 may be provided with the concave / convex pattern 3 having one dimension (for example, about 100 nm) according to the dimension of the object to be separated, or a plurality of different dimensions (for example, 100 nm, 200 nm and 300 nm) may be provided.

  In the former, among the separated objects included in the separated object mixture, only the separated objects having substantially the same dimensions as the uneven pattern 3 can be selectively separated. That is, it is possible to selectively separate a separation object having a size of about 100 nm among the separation objects included in the separation object mixture.

  On the other hand, in the latter, the to-be-separated body according to each dimension of the uneven | corrugated pattern 3 can be isolate | separated among the to-be-separated bodies contained in a to-be-separated body mixture. In the latter case, as shown in FIG. 2, the main surface 21 of the base material portion 2 is divided into a plurality of pattern regions 211 to 213, and uneven patterns 3 having different dimensions are provided in the pattern regions 211 to 213. May be. For example, the pattern region 211 may be provided with a concavo-convex pattern 3 having a size of 100 nm, the pattern region 212 may be provided with a concavo-convex pattern 3 having a size of 200 nm, and the pattern region 213 may be provided with a concavo-convex pattern 3 having a size of 300 nm. In the case of the separated object separating substrate 1 having the structure shown in FIG. 2, a plurality of separated objects having different dimensions are selectively separated in each pattern region 211 to 213 according to the dimensions by one operation. be able to.

  The surface of the concavo-convex pattern 3 may be appropriately subjected to a surface treatment according to the properties of the object to be separated, the material constituting the substrate 2, and the like. For example, membrane vesicles (exosomes), organelles (organelles), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), granules (melanosomes, microbes, etc.) secreted from living cells or cultured cells Body, glyoxisome, bibel-parade body, etc.), cytoskeleton, central body, flagella, cilia, ribosome, virus and the like, it is known that their surfaces show hydrophilicity. Therefore, by subjecting the surface of the concavo-convex pattern 3 to a hydrophilic surface (for example, application of a hydrophilic surface treatment agent), an object to be separated (in vivo cells or cultured cells) that easily adhere to the hydrophilic surface. Secreted membrane vesicles (exosomes), organelles (organelles), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), granules (melanosomes, microsomes, glyoxysomes, bibel parade bodies, etc.) , Cytoskeleton, centriole, flagella, cilia, ribosome, virus, etc.) can be easily and selectively separated. In addition, when using quartz glass as a material which comprises the base material 2, since the surface of the uneven | corrugated pattern 3 is a hydrophilic surface, it is not necessary to perform the said surface treatment.

  In addition, when the surface of the uneven | corrugated pattern 3 is a hydrophilic surface, you may perform the process which makes surfaces other than the surface of the uneven | corrugated pattern 3 among the main surfaces 21 of the base material 2 into a hydrophobic surface. Thereby, it becomes easy to attach the to-be-separated body which adheres easily to a hydrophilic surface by the uneven | corrugated pattern 3. FIG.

  According to the separation target separating substrate 1 according to the present embodiment having the above-described configuration, the concave / convex pattern 3 can be obtained simply by bringing the concave / convex pattern 3 into contact with the separation target mixture containing the fine particulate separation target. Therefore, it is possible to selectively separate the object to be separated according to the dimensions, and therefore it is possible to selectively separate the object to be separated according to the dimension (size). In addition, if the substrate for separation to be separated 1 according to the present embodiment has the concave and convex pattern 3 having substantially the same size as the size of one separated body, the separated body in the form of fine particles is obtained. The effect that it can isolate | separate not as the aggregate | assembly but as an individual can also be show | played. In particular, to-be-separated bodies are membrane vesicles (exosomes), organelles (organelles), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), granules (melanosomes, Microscopic bodies, glyoxisomes, bibel-parade bodies, etc.), cytoskeletons, central bodies, flagella, cilia, ribosomes, viruses, etc., they can be separated as individuals according to their dimensions (size) Therefore, when abnormality (heterogeneity) occurs in the living body-related microparticles, the difference can be analyzed individually as compared with normal living body-related microparticles.

  The substrate separation substrate 1 having the above-described configuration can be manufactured, for example, as follows. FIG. 3 is a process flow diagram showing the manufacturing process of the separation target substrate 1 according to this embodiment in a cut end view.

  As shown in FIG. 3A, first, a base material 2 including a hard mask layer 30 is prepared, a resist film 40 is formed on the hard mask layer 30 of the base material 2, and an electron beam drawing apparatus or the like is used. Then, a pattern latent image is formed on the resist film 40.

  As the base material 2, the constituent material of the substrate for separating object 1 according to the present embodiment (for example, quartz glass, synthetic quartz glass, soda glass, fluorite, calcium fluoride, magnesium fluoride, acrylic glass, borosilicate acid) A substrate made of a glass material such as glass; a transparent material such as a resin material such as polycarbonate, polypropylene, polyethylene, polystyrene, and other polyolefins) can be used.

  The hard mask layer 30 is a layer for forming a hard mask pattern 31 used for etching the base material 2 in a process described later (see FIG. 3D). Therefore, it is comprised with the material which considered the etching selectivity with the material which comprises the base material 2. FIG. For example, when the base material 2 is made of quartz glass, the hard mask layer 30 is preferably made of metal chromium or the like.

  The resist material constituting the resist film 40 is not particularly limited, and a conventionally known energy beam sensitive resist material (for example, an electron beam sensitive resist material, an ultraviolet sensitive resist material, etc.) or the like may be used. it can.

  The pattern latent image formed on the resist film 40 is formed with a dimension (about 10 to 500 nm) substantially the same as the dimension of the concavo-convex pattern 3 on the separation target substrate 1.

  Next, as shown in FIG. 3B, the resist film 40 is developed to form a resist pattern 41. As shown in FIG. 3C, the hard mask layer 30 is formed using the resist pattern 41 as a mask. The hard mask pattern 31 is formed by etching.

  Thereafter, as shown in FIG. 3D, the base material 2 is etched using the hard mask pattern 31 as a mask to form the uneven pattern 3 on the main surface 21 of the base material 2, and finally the hard mask pattern 31 is peeled off. By doing so, as shown in FIG. 3 (e), it is possible to manufacture the substrate for separating object 1 having the base material 2 and the concavo-convex pattern 3 formed on the main surface 21 of the base material 2.

  The substrate for separation to be separated 1 having the above-described configuration makes a separated body having a size substantially the same as the size of the concavo-convex pattern 3 by bringing the concavo-convex pattern 3 into contact with a mixture to be separated including the separation target. The present invention can be applied to a method for separating an object to be separated for selective separation.

  As a method for bringing the concave / convex pattern 3 of the separation target substrate 1 into contact with the separation target mixture containing the separation target, for example, the separation target separation substrate 1 is placed in a container containing the separation target mixture. For example, a method of placing the concavo-convex pattern 3 and the object to be separated (mixture for separation), a method for causing the mixture to be separated to flow on the concavo-convex pattern 3 of the substrate for separation-to-be-separated 1 placed tilted, etc. Is mentioned.

  In the method of placing the separation target separation substrate 1 in the container for containing the separation target mixture and bringing the concave / convex pattern 3 and the separation target into contact with each other, the time for contacting the concave / convex pattern 3 and the separation target is as follows: There is no particular limitation as long as it is a sufficient time for selectively separating the separated object.

  In addition, as long as it has predetermined fluidity | liquidity, the to-be-separated material mixture in this embodiment is the liquid (For example, suspension containing a to-be-separated object of microparticulates, as a to-be-separated object) Membrane vesicles (exosomes), organelles (organelles), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), granules (melanosomes, microsomes, glyoxysomes, bibel parade bodies, etc.), cytoskeleton In vivo body fluids and cell culture fluids containing biologically relevant microparticles such as centriole, flagella, cilia, ribosomes, viruses, etc.), colloids using a liquid as a dispersion medium may be used. .

  As described above, by using the separation target separating substrate 1 according to the present embodiment, a separation target having substantially the same size as the uneven pattern 3 is obtained from the separation target mixture including the separation target. It can be selectively separated. Moreover, since the uneven | corrugated pattern 3 of the to-be-separated object isolation | separation board | substrate 1 and the to-be-separated object (to-be-separated object mixture) can only be contacted, the to-be-separated object of a desired dimension can be selectively isolate | separated, The separation object can be separated without applying a load to the separation object as in the conventional separation method using centrifugal force. Therefore, there is an effect that separation can be performed without damaging the object to be separated.

  The embodiment described above is described for facilitating understanding of the present invention, and is not described for limiting the present invention. Therefore, each element disclosed in the above embodiment is intended to include all design changes and equivalents belonging to the technical scope of the present invention.

  In the said embodiment, although the board | substrate 1 for to-be-separated object which has the uneven | corrugated pattern 3 formed in the main surface 21 of the base material 2 as an integral uneven structure was mentioned as an example, this invention is such It is not limited to the embodiment. For example, the separation target substrate 1 has a concavo-convex structure (for example, a sheet having the concavo-convex pattern 3) having the concavo-convex pattern 3 configured as a member separate from the base 2 on the main surface 21 of the base 2. A member etc. may be attached.

  In the said embodiment, although the uneven | corrugated pattern 3 is formed by etching and the hard mask pattern 31 is peeled after that, the to-be-separated body isolation | separation board | substrate 1 is manufactured, This invention is limited to such an aspect. It is not a thing. For example, the base material 2 on which the uneven pattern 3 is formed may be thinned without changing the size and height of the uneven pattern 3. Moreover, you may manufacture the to-be-separated object isolation | separation board | substrate 1 by forming the uneven | corrugated pattern 3 in the several area | region on the base material 2, and cut | disconnecting (dividing into pieces) for every area | region.

  In the above embodiment, as a method of separating the separated object using the separated object separating substrate 1, the separated object separating substrate 1 is placed in a container containing the separated object mixture, and the uneven pattern 3 And a separation method using a method of bringing a separation target (mixture to be separated) into contact with each other, a method of allowing the mixture to be separated to flow on the concavo-convex pattern 3 of the substrate for separation to be separated 1 placed at an inclination, etc. However, the present invention is not limited to such an embodiment. For example, in the separation method of the separated object using the separated object separating substrate 1, the uneven pattern 3 and the separated object are brought into contact with each other, and the separated object is separated using the action of centrifugal force. It may be a method.

  EXAMPLES Hereinafter, although an Example etc. are given and this invention is demonstrated further in detail, this invention is not limited at all by the following Example etc.

[Production Example of Substrate Separating Substrate 1]
A quartz glass substrate is prepared as a base material 2 on which a hard mask layer 30 made of Cr having a thickness of 6 nm is provided on the main surface 21, and an electron beam sensitive resist (product name: SEBP-9012, manufactured by Shin-Etsu Chemical Co., Ltd.). ) Was applied onto the hard mask layer 30 to form a resist film 40. A pillar-shaped resist pattern 41 (dimension: 50 nm) was formed on the resist film 40 using an electron beam drawing apparatus.

Next, the hard mask layer 30 was dry-etched (etching gas: Cl ₂ + O ₂ ) using the resist pattern 41 as a mask, and the remaining resist pattern 41 was removed to form the hard mask pattern 31.

  The quartz glass substrate 2 was etched using the hard mask pattern 31 formed as described above as a mask, and a pillar-shaped uneven pattern 3 having a dimension of 50 nm was formed on the main surface 21 of the quartz glass substrate 2. Finally, the hard mask pattern 31 was peeled off to produce the separation target substrate 1 (Sample 1).

  Further, the dimensions of the resist pattern 41 are changed to 100 nm, 200 nm, 250 nm, and 300 nm, respectively, and the pillar-shaped uneven pattern 3 having dimensions of 100 nm, 200 nm, 250 nm, and 300 nm is formed on the main surface 21 of the quartz glass substrate 2 respectively. Manufactured the to-be-separated object isolation | separation board | substrate 1 like the above (samples 2-5).

[Example 1]
Five containers containing the supernatant of the stem cell culture solution (mixture to be separated) are prepared, and the separate substrates 1 (samples 1 to 5) manufactured according to the above production examples are placed in the respective containers. Then, the supernatant liquid was brought into contact with the separation target substrate 1. After elapse of a predetermined time, each separation target separating substrate 1 was taken out, and the top of the uneven pattern 3 was observed using an optical microscope, a laser microscope, and a scanning electron microscope. As a result, it was confirmed that 30 to 60 nm membrane vesicles adhered to the uneven pattern 3 (dimension: 50 nm) of the substrate 1 for separation of the sample 1. In addition, it was confirmed that 80 to 120 nm of membrane vesicles were attached on the uneven pattern 3 (dimension: 100 nm) of the substrate separation target substrate 1 of the sample 2. Further, it was confirmed that 160 to 240 nm membrane vesicles were adhered on the uneven pattern 3 (dimension: 200 nm) of the separation target substrate 1 of the sample 3. Furthermore, it was confirmed that 200 to 300 nm of membrane vesicles were adhered on the uneven pattern 3 (dimension: 250 nm) of the separation target substrate 1 of the sample 4. In addition, it was confirmed that 240 to 360 nm membrane vesicles were adhered on the uneven pattern 3 (dimension: 300 nm) of the substrate 5 to be separated of the sample 5.

  As a result of this example, according to the substrate for separating objects 1 to be separated, it is possible to selectively separate a separated object having a size corresponding to the size of the concavo-convex pattern 3 from the separated object mixture including the separated object. It became clear that there was.

  INDUSTRIAL APPLICABILITY The present invention is useful in a technical field that requires selective separation of microparticulates to be separated, particularly in a technical field that selectively separates and analyzes biologically relevant microparticles such as membrane vesicles (exosomes).

DESCRIPTION OF SYMBOLS 1 ... Substrate isolation | separation board | substrate 2 ... Base material part 21 ... Main surface 211-213 ... Pattern area | region 3 ... Concave-convex pattern 31 ... Concave part 32 ... Convex part

Claims (6)

A method of separating the separation object from a separation object mixture containing a fine particle separation object,
The separation object is attached to the concavo-convex pattern by bringing the separation object mixture into contact with a concavo-convex structure having a concavo-convex pattern including a concave portion and a convex portion having a dimension of 10 to 500 nm, and the separation subject is Separating from the mixture to be separated;
The uneven pattern may have a said object separator substantially the same dimensions,
The separation object is a membrane vesicle secreted from a living cell or a cultured cell,
The uneven structure, the method of separating the separator characterized that you have been composed of quartz glass. Of the concavo-convex structure, a region in contact with the separated mixture is divided into a plurality of regions,
2. The method for separating an object to be separated according to claim 1, wherein dimensions of the concavo-convex pattern provided in each of the plurality of regions are different from each other.   The method for separating a material to be separated according to claim 1 or 2, wherein the surface of the concavo-convex structure that comes into contact with the material to be separated is a hydrophilic surface. A substrate for separating material to be separated used for separating the material to be separated from a material mixture to be separated containing a fine particle-shaped material to be separated,
A substrate and a concavo-convex pattern including a concave portion and a convex portion having a dimension of 10 to 500 nm formed on one surface of the base material,
The concavo-convex pattern has substantially the same dimensions as the separated object,
The separation object is a membrane vesicle secreted from a living cell or a cultured cell,
The substrate is made of quartz glass;
The separation target mixture can be separated from the separation target mixture by bringing the separation target mixture into contact with the concavo-convex pattern by bringing the separation target mixture into contact with the separation target separation substrate. A substrate for separating objects to be separated. The concavo-convex pattern is formed in each of a plurality of regions formed by dividing one surface of the base material,
The substrate for separating objects according to claim 4 , wherein dimensions of the concave portion and the convex portion of the concave / convex pattern provided in each of the plurality of regions are different from each other. The substrate for separating objects according to claim 4 or 5 , wherein the surface of the concave-convex pattern is a hydrophilic surface.

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