Substrate for collecting minute objects and method for manufacturing the same

Abstract

To provide a substrate for collecting minute objects that can retain air, which serves as a core of generation of microbubbles in pores, and to provide a method for manufacturing the same.SOLUTION: A large number of pores 12a capable of retaining air are formed on a surface of a collection substrate 10. These pores 12a are formed in recesses in a resist film 12 formed as a porous film on a surface of a glass substrate 11 serving as a base of the collection substrate 10. A thin film 13 is formed on a head part of the resist film 12 and a bottom surface of the pore 12a. The thin film 13 forms a photothermal conversion region that converts laser beams L1 to L9 irradiated from laser light sources 41 to 49 into heat. A water-repellent film 14 is formed on the surface of the collection substrate 10 covering the thin film 13.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a collection substrate for collecting a plurality of minute objects dispersed in a liquid, and a method for manufacturing the same. [Background technology]

[0002] Conventionally, as a collection substrate for this type of minute object, there is, for example, one disclosed as an integrated kit in FIG. 20 of Patent Document 1.

[0003] This accumulation kit consists of a substrate, a honeycomb polymer film, and a thin film, and a liquid containing microscopic bacteria is dropped onto the honeycomb polymer film. A cover glass is used for the substrate, and a resin is used for the honeycomb polymer film. The thin film is made of a material that absorbs laser light and converts light energy into thermal energy. When the thin film is irradiated with light rays and the liquid is heated, microbubbles are generated at the position where the light rays are irradiated. The generated microbubbles cause convection in the liquid in a specific direction, and multiple microscopic objects accumulate in the area near the microbubbles. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2020 / 218347 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the conventional micro-object collection substrate described above, when a liquid containing micro-objects is dropped onto the substrate, water penetrates into the pores of the honeycomb polymer membrane, which is a porous membrane. Therefore, the air that serves as the nucleus for generating microbubbles, which is necessary for collecting micro-objects, cannot be retained within the pores, or, even if it is retained, it is quickly released and defoamed. Therefore, in order to newly generate the air that serves as the nucleus for generating microbubbles, it is necessary to irradiate the thin film, which is the photothermal conversion region, with high-intensity laser light. As a result, the micro-object collection device lacks convenience in use.

[0006] In order to solve these problems, the present invention aims to provide a substrate for collecting micro-objects that can retain air, which is the nucleus for generating microbubbles, within its pores, and a method for manufacturing the same. [Means for solving the problem]

[0007] To this end, the present invention provides a porous film formed on the surface of the substrate and having a plurality of pores capable of retaining air; a photothermal conversion region formed on the surface of the substrate covering the porous film, which converts light irradiated from the light source into heat; a water-repellent film having water repellency formed on the surface of the substrate covering the photothermal conversion region; A micro-object collection substrate was constructed to collect a plurality of micro-objects dispersed in a liquid in which the porous membrane is immersed.

[0008] According to this configuration, because the substrate surface covering the photothermal conversion region is provided with a water-repellent film, even when a liquid containing micro-objects is dropped onto the substrate, the water-repellent film prevents the liquid from penetrating the pores of the porous film, and air is retained within the pores of the porous film. This eliminates the need to newly generate air, which serves as the nucleus for microbubble generation, as in the past, and makes it possible to capture micro-objects near the microbubbles without increasing the intensity of the laser light irradiated onto the photothermal conversion region. As a result, the convenience of using the micro-object collection device is improved.

[0009] The present invention also provides forming a porous film having a plurality of pores capable of retaining air on a surface of a substrate; forming a photothermal conversion region that converts light irradiated from a light source into heat on the surface of the substrate that covers the porous film; and exposing the substrate surface to vapor of a silane coupling agent and water vapor for a predetermined time in an atmosphere at a predetermined temperature to form a water-repellent film on the substrate surface covering the photothermal conversion region. A method for manufacturing a micro-object collection substrate for collecting a plurality of micro-objects dispersed in a liquid in which a porous membrane is immersed has been developed.

[0010] According to this configuration, by exposing the substrate surface to vapor of a silane coupling agent and water vapor in an atmosphere at a predetermined temperature for a predetermined time, the hydrophilic groups on the surface of the photothermal conversion region are replaced with hydrophobic groups, forming a water-repellent film on the substrate surface covering the photothermal conversion region. Therefore, as described above, this water-repellent film prevents the liquid from penetrating the pores of the porous film when a liquid containing micro-objects is dropped onto the substrate, thereby maintaining air within the pores. Therefore, this configuration also eliminates the need to newly generate air, which serves as the nucleus for microbubble generation, as in the conventional method. This makes it possible to capture micro-objects near the microbubbles without increasing the intensity of the laser light irradiated onto the photothermal conversion region. As a result, the convenience of using the micro-object collection device is improved. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a substrate for collecting minute objects that can retain air, which serves as the nucleus for generating microbubbles, within the pores, and a method for manufacturing the same. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a block diagram showing a schematic configuration of a minute-object collection device configured using a minute-object collection substrate according to an embodiment of the present invention. [Figure 2](a) is a partially enlarged cross-sectional side view illustrating the state of the collection substrate placed on the XYZ-axis stage that constitutes the micro-object collection device shown in Figure 1, and (b) is a diagram illustrating the generation of microbubbles when light is irradiated onto the collection substrate shown in (a). [Figure 3] FIG. 1A is a perspective view of a collection substrate for minute objects according to an embodiment, and FIG. 1B is a side cross-sectional view. [Figure 4] 5(a) to 5(e) are cross-sectional views of a collection substrate in each step of a method for manufacturing a collection substrate for minute objects according to one embodiment of the present invention. [Figure 5] 10 is a graph showing the heat conversion efficiency due to the photothermal effect of a gold thin film formed on a minute object collection substrate according to one embodiment. [Figure 6] FIG. 10 is a partially enlarged cross-sectional side view of a collection substrate according to one embodiment, illustrating that the contact angle of the substrate surface with water increases when the substrate surface is treated with a silane coupling agent. [Figure 7] (a) is a plan view of a collection substrate for explaining the irradiation area of ​​laser light irradiated onto the collection substrate for micro-objects in one embodiment, and (b) is a plan view of a collection substrate for explaining an example in which the irradiation area of ​​laser light is formed in one location. [Figure 8] 10 is a plan view showing a modified example of a region where pores are formed on the surface of a collection substrate for minute objects according to an embodiment of the present invention. FIG. [Figure 9] 10(a) to 10(c) are cross-sectional views of a collection substrate illustrating a modified example of the method for producing a porous membrane in the method for producing a collection substrate for minute objects according to one embodiment. [Figure 10] 10(a) and 10(b) are table diagrams showing the results of a demonstration experiment in which the generation of microbubbles MB in water was confirmed when water was poured onto a collection substrate manufactured according to a method for manufacturing a collection substrate for micro-objects in one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0013] Next, an embodiment for carrying out the minute object collection substrate and the method for manufacturing the same of the present invention will be described.

[0014] In the present invention and its embodiments, the term "micro object" refers to an object having a size ranging from the order of nanometers to the order of micrometers. The shape of the micro object is not particularly limited, and may be, for example, a sphere, an ellipsoid, or a rod. When the micro object is an ellipsoid, at least one of the length in the major axis direction and the length in the minor axis direction of the ellipsoid may be in the order of nanometers to the order of micrometers. When the micro object is a rod, at least one of the width and length of the rod may be in the order of nanometers to the order of micrometers.

[0015] The minute objects may include objects of biological origin. More specifically, the minute objects may include, for example, cells, microorganisms (bacteria, fungi, etc.), antigens (allergens, etc.), viruses, and biological materials. "Biological materials" may include biopolymers such as proteins, nucleic acids, lipids, and polysaccharides.

[0016] Other examples of microscopic objects include metal nanoparticles, metal nanoparticle aggregates, metal nanoparticle assembly structures, semiconductor nanoparticles, organic nanoparticles, resin beads, and particulate matter (PM). A "metal nanoparticle" is a metal particle with a size on the order of nanometers. A "metal nanoparticle aggregate" is an aggregate formed by the aggregation of multiple metal nanoparticles. A "metal nanoparticle assembly structure" is a structure in which multiple metal nanoparticles are fixed to the surface of a bead via interaction sites, with gaps between them and spaced apart at intervals less than the diameter of the metal nanoparticle. A "semiconductor nanoparticle" is a semiconductor particle with a size on the order of nanometers. An "organic nanoparticle" is a particle made of an organic compound with a size on the order of nanometers. A "resin bead" is a particle made of resin with a size on the order of nanometers to micrometers. A "PM" is a particulate matter with a size on the order of micrometers.

[0017] Furthermore, in the present invention and its embodiments, "nanometer order" includes a range of 1 nm to 1000 nm (= 1 μm). "Micrometer order" includes a range of 1 μm to 1000 μm (= 1 mm). Therefore, the term "range from nanometer order to micrometer order" includes a range of 1 nm to 1000 μm. The term "range from nanometer order to micrometer order" typically indicates a range of several nm to several hundred μm, preferably a range of 100 nm to 100 μm, and more preferably a range of 1 μm to several tens of μm.

[0018] In the present invention and its embodiments, a "pore" refers to a hole having an opening ranging from the order of nanometers to the order of micrometers. The shape of the pore is not particularly limited and may include any shape such as a cylinder, a prism, or a sphere other than a perfect sphere (e.g., a hemisphere or a semi-ellipsoid).

[0019] In the present invention and its embodiments, "microbubbles" refer to air bubbles on the order of micrometers.

[0020] 1 is a block diagram showing the schematic configuration of a minute object collection device 1 configured using a minute object collection substrate according to one embodiment of the present invention. Note that in the following figures, the same or corresponding parts are given the same reference numerals, and overlapping explanations will be omitted.

[0021] The collection device 1 includes a collection substrate 10, an XYZ-axis stage 20, an adjustment mechanism 30, laser light sources 41-49, optical components 50, an objective lens 60, an illumination device 70, an imaging device 80, a sample supply device 90, and a control device 100. Hereinafter, the x and y directions represent horizontal directions. The x and y directions are perpendicular to each other. The z direction represents the vertical direction. The direction of gravity is downward in the z direction.

[0022] The collection substrate 10 holds the sample S. In this embodiment, the sample S is a liquid in which minute objects α are dispersed. The detailed configuration of the collection substrate 10 will be described with reference to FIG. 3. The collection substrate 10 is placed on an XYZ axis stage 20. The sample supply device 90 supplies the liquid sample S by dropping it onto the collection substrate 10 in response to a command from the control device 100. A dispenser, for example, can be used as the sample supply device 90.

[0023] The adjustment mechanism 30 adjusts the position of the XYZ-axis stage 20, on which the collection substrate 10 is mounted, in the x, y, and z directions in response to commands from the control device 100. In this embodiment, the position of the objective lens 60 is fixed, so the relative positional relationship between the collection substrate 10 and the objective lens 60 is adjusted by adjusting the position of the XYZ-axis stage 20. As the adjustment mechanism 30, for example, a driving mechanism such as a servo motor and a focusing handle attached to the microscope can be used, but the specific configuration of the adjustment mechanism 30 is not particularly limited. The adjustment mechanism 30 may also adjust the position of the objective lens 60 with respect to the fixed collection substrate 10.

[0024] The nine laser light sources 41 to 49 emit, for example, near-infrared laser light L1 to L9 (for example, wavelength 850 nm) in response to commands from the control device 100. However, the wavelengths of the laser light L1 to L9 are not limited to this, as long as they are within the light absorption band of the material of the thin film 13 (see FIG. 3) described below.

[0025] Optical component 50 includes, for example, a mirror, a dichroic mirror, or a prism. The optical system of collection device 1 is adjusted so that laser light L1 to L9 from laser light sources 41 to 49 is guided to objective lens 60 by optical component 50.

[0026] The objective lens 60 collects the laser beams L1 to L9 emitted from the laser light sources 41 to 49. The light collected by the objective lens 60 is irradiated onto the collection substrate 10. Here, "irradiate" includes the case where the laser beams L1 to L9 pass through the collection substrate 10. In other words, it is not limited to the case where the beam waist of the light collected by the objective lens 60 is located inside the collection substrate 10. The optical component 50 and the objective lens 60 can be incorporated into, for example, an inverted microscope body or an upright microscope body.

[0027] In response to a command from the control device 100, the lighting device 70 emits white light WL to illuminate the sample S dropped on the collection substrate 10. As one example, a halogen lamp can be used for the lighting device 70. The objective lens 60 is also used to capture the white light WL irradiated from the lighting device 70 onto the collection substrate 10. The white light WL captured by the objective lens 60 is guided to the photographing device 80 by the optical component 50.

[0028] In response to a command from the control device 100, the photographing device 80 photographs the sample S (see FIG. 2) on the collection substrate 10 irradiated with the white light WL, and outputs the photographed image to the control device 100. The photographing device 80 is a video camera including a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor.

[0029] The control device 100 controls the adjustment mechanism 30, the laser light sources 41 to 49, the lighting device 70, the photographing device 80, and the sample supplying device 90. The control device 100 also performs predetermined image processing on the image captured by the photographing device 80. The control device 100 is realized by a microcomputer including a CPU (Central Processing Unit), a memory, an input / output buffer, etc., all of which are not shown.

[0030] 1 and may include optical fibers or the like, as long as the optical system of collection device 1 is capable of irradiating collection substrate 10 with laser beams L1 to L9 from laser light sources 41 to 49 and capturing white light WL from collection substrate 10 into photographing device 80. Furthermore, in collection device 1, lighting device 70 and photographing device 80 are merely devices for photographing the state of sample S and are not essential components for collection of minute objects by collection device 1. Furthermore, nine laser light sources 41 to 49 are not necessarily required, and at least one will suffice.

[0031] 2(a) is a partially enlarged side cross-sectional view illustrating the state of the collection substrate 10 placed on the XYZ axis stage 20. The collection substrate 10 is covered with a sample S containing dispersed micro-objects α, which is dropped from a sample supplying device 90. The collection substrate 10 collects the multiple micro-objects α dispersed in the liquid of the sample S.

[0032] FIG. 3(a) is a perspective view showing a schematic configuration of the collection substrate 10, and FIG. 3(b) is a side cross-sectional view. A large number of cylindrical pores 12a capable of retaining air are formed on the surface of the collection substrate 10. As shown in FIG. 3(b), these pores 12a are formed in recesses of a resist film 12 formed as a porous film on the surface of a glass substrate 11 that serves as the base of the collection substrate 10. The resist film 12 has a plurality of pores 12a. A thin film 13 is formed on the top of the resist film 12 and on the bottom surfaces of the pores 12a. The thin film 13 constitutes a photothermal conversion region that converts laser beams L1 to L9 irradiated from laser light sources 41 to 49 into heat. The laser light sources 41 to 49 constitute light sources that irradiate the collection substrate 10 with light. A water-repellent film 14 is formed on the surface of the collection substrate 10 that covers the thin film 13.

[0033] In this embodiment, the collection substrate 10 is formed using photolithography technology as shown in Fig. 4. Figures 4(a) to 4(e) are cross-sectional views of the collection substrate 10 during each step of a method for manufacturing the collection substrate 10 for minute objects α according to one embodiment of the present invention.

[0034] First, as shown in FIG. 1A, a resist film 12 is applied to a light-transmitting glass substrate 11 made of tempac glass. In this embodiment, SU-8 is used as the material for the resist film 12. Next, a photomask 15 is formed on the resist film 12, and the photomask 15 is patterned as shown in FIG. 1B. Next, the resist film 12 is selectively hardened by exposure using the photomask 15, and then the photomask 15 and the unhardened resist film 12 are removed. As a result, a plurality of pores 12a are selectively formed on the glass substrate 11 in the areas where the resist film 12 is not present, as shown in FIG. 1C.

[0035] Then, a thin film 13 is formed on the surface of the collection substrate 10, covering the resist film 12, by vapor deposition, sputtering, electroless plating, or other techniques. As shown in FIG. 1(d), the thin film 13 is formed on the bottom surfaces of the pores 12a and on the upper surface of the resist film 12. The material of the thin film 13 is preferably a material with high light absorption (e.g., photothermal conversion efficiency) in the wavelength band of the laser beams L1 to L9 (near-infrared band in this embodiment). In this embodiment, a gold (Au) thin film with a thickness on the order of nanometers is used as the material for the thin film 13, and is formed by vapor deposition. However, it may be made of a metal element other than gold (e.g., silver) or a metal nanoparticle assembly structure (e.g., a structure using gold nanoparticles or silver nanoparticles). Alternatively, it may be a non-metallic material with high light absorption in the wavelength band of the laser beams L1 to L9. Examples of such materials include a material similar to a blackbody (e.g., a carbon nanotube blackbody).

[0036] Next, the surface of the collection substrate 10 is exposed to vapor of a silane coupling agent and water vapor in an atmosphere at a predetermined temperature for a predetermined time, and as shown in FIG. 1(e), a water-repellent film 14 having water repellency is formed on the substrate surface covering the thin film 13. In detail, in this step in the present embodiment, first, the surface of the collection substrate 10 covered with the thin film 13 is cleaned with ethanol and water. Next, the surface of the collection substrate 10 is activated by UV (ultraviolet) irradiation treatment. Subsequently, the collection substrate 10 is exposed to an atmosphere containing vapor of the silane coupling agent and water vapor at 150°C for one hour. Thereafter, unreacted silane coupling agent remaining on the surface of the collection substrate 10 is removed with ethanol and water, and the collection substrate 10 is dried, and as shown in FIG. 1(e), a water-repellent film 14 is formed on the substrate surface covering the thin film 13.

[0037] By subjecting the substrate surface to such surface treatment using a silane coupling agent, hydrophilic groups such as OH groups present on the surface of the thin film 13 are replaced with hydrophobic groups such as CH 3 , thereby forming a water-repellent film 14 .

[0038] Although it is possible to form a water-repellent film by fluorine-treating the substrate surface, the water-repellent film 14 formed by treatment with a silane coupling agent is a film with a molecular-level thickness and therefore does not block the pores 12a. Furthermore, since the water-repellent film 14 transmits light, it becomes possible to observe the minute object α through the water-repellent film 14 from the bottom side of the glass substrate 11 after the minute object α is collected.

[0039] 5 is a graph showing the heat conversion efficiency due to the photothermal effect of the thin film 13 constituting the photothermal conversion region. The horizontal axis of the graph represents the thickness [nm] of the gold thin film used as the thin film 13, and the vertical axis represents the substrate temperature [°C] of the collection substrate 10.

[0040] As described below, when the thin film 13 is irradiated with the laser beams L1 to L9, heat is generated at the irradiated points on the collection substrate 10, generating microbubbles MB. However, from the graph, it can be seen that when the gold thin film is 4 nm or less in thickness, the laser beams L1 to L9 pass through the thin film 13, reducing the heat conversion efficiency and preventing the generation of microbubbles MB. Furthermore, when the gold thin film is 20 nm or more in thickness, the thin film 13 has high thermal conductivity, reducing the heat conversion efficiency and preventing the generation of microbubbles MB. For this reason, the thin film 13 is preferably formed from a gold thin film with a thickness of 4 nm to 20 nm.

[0041] By forming the thin film 13 from a gold thin film with a thickness of 4 nm or more, it is possible to avoid a situation in which the laser beams L1 to L9 irradiated from the laser light sources 41 to 49 pass through the gold thin film, reducing the heat conversion efficiency and preventing the generation of microbubbles MB. Also, by forming the thin film 13 from a gold thin film with a thickness of 20 nm or less, it is possible to avoid a situation in which the thermal conductivity of the thin film 13 becomes too high, reducing the heat conversion efficiency and preventing the generation of microbubbles MB.

[0042] In this embodiment, as shown in FIG. 3(a), a resist film 12 is formed on a glass substrate 11 that is a square with sides of 5 mm in plan view and has a thickness of 0.2 mm. A thin film 13 is evaporated to a target thickness of 10 nm. The pores 12a are formed in a cylindrical shape with a diameter φ of 5 μm and a depth of 5 μm, and are arranged at a pitch of 10 μm in the x and y directions. Each pore 12a of this size is capable of retaining air 16 in the liquid of the sample S, as shown in FIG. 2.

[0043] Furthermore, from the viewpoint of photolithography technology, it is desirable that the pores 12a have a cylindrical shape. However, in order to store air, it is desirable that the pores 12a have a substantially cylindrical shape with inverted tapered side surfaces that make the opening area smaller than the bottom area, as shown in the partially enlarged side cross-sectional view of Figure 6. With this configuration, the pores 12a have an inverted tapered vertical cross-sectional shape, which makes it more difficult for the air 16 stored in the pores 12a to escape.

[0044] 7(a) is a plan view of the collection substrate 10 of this embodiment, in which irradiation regions (hereinafter referred to as laser spots) LS1 to LS9 onto which laser beams L1 to L9 are irradiated are indicated by black dots. In this embodiment, the laser spots LS1 to LS9 are formed at nine locations on the thin film 13 on the surface of the collection substrate 10. However, the number of laser spots is not limited to nine, and they may be formed at only one location as shown in FIG. 7(b). In this case, if one laser light source 41 is provided instead of the laser light sources 41 to 49, for example, the laser spot LS1 is formed at one location.

[0045] When collecting a plurality of minute objects α dispersed in a sample S immersed in a resist film 12 using the collection substrate 10, the control device 10 first places the collection substrate 10 on the XYZ-axis stage 20. This process is achieved, for example, by a feeding mechanism (not shown) for the collection substrate 10. Next, the control device 100 controls the sample supply device 90 to drop the sample S onto the collection substrate 10 placed on the XYZ-axis stage 20. Next, the control device 100 controls the illumination device 70 to emit white light WL to irradiate the sample S on the collection substrate 10, and controls the imaging device 80 to begin imaging the sample S.

[0046] Next, the control device 100 adjusts the position of the XYZ-axis stage 20 by controlling the adjustment mechanism 30 so that the laser beams L1 to L9 from the laser light sources 41 to 49 are irradiated onto appropriate positions on the collection substrate 10, for example, positions where the laser spots LS1 to LS9 in Fig. 7(a) are formed. This position adjustment can be achieved by extracting the pattern of the pores 12a from the image captured by the imaging device 80, for example, using an image processing technique for pattern recognition.

[0047] Next, the control device 100 controls the laser light sources 41 to 49 to emit laser beams L1 to L9. The laser beams L1 to L9 are focused by the objective lens 60, and the focused beams are irradiated onto the thin film 13 of the collection substrate 10. This causes convection in the liquid of the sample S, and the minute objects α dispersed in the liquid are collected near the laser spots LS1 to LS9 and trapped in the pores 12a. Thereafter, the control device 100 controls the laser light sources 41 to 49 to stop irradiating the thin film 13 of the collection substrate 10 with the laser beams L1 to L9.

[0048] The manner in which the minute particles α are collected and the mechanism thereof will be explained as follows.

[0049] FIG. 2(b) shows how minute objects α are collected from the sample S when a laser spot LS1 is formed at one location in the center of the collection substrate 10 as shown in FIG. 7(b).

[0050] Before irradiation of the laser light L1 from the laser light source 41 begins, the minute objects α can move freely in the liquid of the sample S, as shown in FIG. 2(a). Almost no minute objects α are trapped in the pores 12a of the collection substrate 10. However, when irradiation of the laser light L1 (hereinafter also abbreviated as "light irradiation") begins, the vicinity of the laser spot LS1 is locally heated due to the photothermal effect of the thin film 13 of the laser spot LS1. As a result, microbubbles MB are generated at the laser spot LS1.

[0051] At this time, when heat is locally generated in the pores 12a that hold the air 16, microbubbles MB are generated around the air 16 held in the pores 12a, and this generation is accelerated. The locally generated heat increases the temperature of the liquid closer to the light irradiation point, creating a temperature gradient in the liquid. This temperature gradient causes convection in the liquid held on the XYZ axis stage 20, and this convection is efficient because it accelerates the generation of microbubbles MB. Due to this efficiently generated convection, the micro-objects α dispersed in the liquid are efficiently captured by the microbubbles MB formed on the surface of the collection substrate 10 and are then captured in the pores 12a.

[0052] More specifically, a stagnation region where the convection flow velocity is zero occurs between the microbubbles MB and the thin film 13. The minute objects α carried by the convection currents remain in this stagnation region and are captured. Here, "capturing" refers to the action of the minute objects α gathering in the vicinity of the microbubbles MB. As a result, the frequency (number of times per unit time) of the minute objects α passing above the pores 12a around the microbubbles MB increases compared to the case without light irradiation. The minute objects α are captured within the pores 12a as they pass above the pores 12a. Here, "capturing" refers to the action of capturing the minute objects α in the space within the pores 12a.

[0053] In the collection substrate 10 for minute objects α according to this embodiment, the substrate surface covering the thin film 13, which is the photothermal conversion region, is provided with a water-repellent film 14. Therefore, even when a liquid sample S containing minute objects α is dropped onto the collection substrate 10, the water-repellent film 14 prevents the liquid of the sample S from penetrating into the pores 12a, and air 16 continues to be held in the pores 12a of the resist film 12, which is a porous film.

[0054] That is, the material of thin film 13, which is the photothermal conversion region, has a smaller contact angle θ with water than the resin material of resist film 12, but when the substrate surface is treated with a silane coupling agent to form water-repellent film 14, the substrate surface is coated with hydrophobic groups, which are organic functional groups, and the contact angle θ with water becomes larger. Generally, as shown in Figure 6, it is known that when the contact angle θ with liquid 17 on the substrate surface is larger than the angle ψ of the geometric structure of the substrate surface (θ > ψ), the force F acting on the gas-liquid interface between air 16 and liquid 17 is upward, as shown in the figure.

[0055] Therefore, when the water-repellent film 14 is formed and the contact angle θ with water on the substrate surface increases, the interfacial tension of the gas-liquid interface acts upward, preventing water from entering the pores 12a. Therefore, with the collection substrate 10 for minute objects α according to this embodiment, air 16 continues to be held in the pores 12a, eliminating the need to newly generate air that serves as the nucleus for generating microbubbles MB as in the conventional method, and making it possible to collect minute objects α near the microbubbles MB without increasing the intensity of the laser light irradiated on the photothermal conversion region.

[0056] Furthermore, in the method for manufacturing a collection substrate according to the present embodiment, which captures a plurality of minute objects α dispersed in a sample S immersed in a porous resist film 12, the substrate surface is exposed to vapor of a silane coupling agent and water vapor for a predetermined time in an atmosphere at a predetermined temperature, as described with reference to FIG. 4(e). This converts hydrophilic groups on the surface of the thin film 13 into hydrophobic groups, forming a water-repellent film 14 on the substrate surface covering the thin film 13. Therefore, even when the sample S containing the minute objects α is dropped onto the collection substrate 10, the water-repellent film 14 prevents the liquid of the sample S from penetrating the pores 12a, thereby allowing air 16 to be continuously retained within the pores 12a. Therefore, this configuration also eliminates the need to newly generate air, which serves as the nucleus for generating microbubbles MB, as in the conventional method. This makes it possible to capture minute objects α near the microbubbles MB without increasing the intensity of the laser light irradiated onto the photothermal conversion region.

[0057] Therefore, according to the present embodiment, it is possible to provide a minute object collection substrate 10 and a manufacturing method thereof that can retain the air 16, which is the nucleus for generating microbubbles MB, within the pores 12a, thereby improving the convenience of using the collection device 1 for minute objects α.

[0058] In this embodiment, the diameter φ of the pores 12a is 5 μm, but it is preferable that the diameter φ of the pores 12a is 5 μm or less. According to this configuration, it becomes easier to retain the air 16 in the pores 12a.

[0059] In the above embodiment, the case where pores 12a are provided on the entire surface of the collection substrate 10 has been described. However, as in the collection substrate 10A shown in the plan view of Fig. 8, the resist film 12, which is a porous film, may be configured so that multiple pores 12a are formed only in areas where laser spots LS1 to LS9 are formed by irradiation with laser light from the laser light sources 41 to 49. With this configuration, heat is generated in areas where the multiple pores 12a are formed and the areas where the laser spots LS1 to LS9 are formed overlap, and microbubbles MB are selectively formed in those areas. As a result, microbubbles MB become noise bubbles, preventing microbubbles MB from being formed in areas where microbubbles MB are not desired, and preventing dispersion of the minute objects α.

[0060] In the above embodiment, the pores 12a of the porous resist film 12 are formed by photolithography. However, the porous film may also be formed by the Breath Figure method (BF method). In the BF method, condensation is caused in a polymer solution on a substrate, and the regularly aligned condensed water droplets are used as a template to form a polymer honeycomb porous film in a self-organizing manner.

[0061] For example, when an airflow containing water vapor is passed over a polymer solution 18 on a glass substrate 11 shown in FIG. 9(a), condensation occurs on the surface of the polymer solution 18 due to the temperature difference between the polymer solution 18 and the airflow, and multiple water droplet nuclei 19 are generated within the polymer solution 18. Each water droplet nuclei 19 grows over time, and the solvent continues to evaporate. After the solvent is lost, the multiple water droplet nuclei 19 become water droplets 19a, which self-organize into a honeycomb-like arrangement, and the polymer solution 18 becomes a polymer film 18a, and the water droplets 19a are fixed within the polymer film 18a as shown in FIG. 9(b). Subsequently, as each water droplet 19a evaporates, interconnected spherical pores 19b are formed within the polymer film 18a, forming a polymer honeycomb porous film.

[0062] According to the BF method, a porous film having small diameter pores 19b can be easily formed. After the porous film is formed, a photothermal conversion region and a water-repellent film are formed on the porous film in the same manner as in the above embodiment.

[0063] Alternatively, the porous film may be formed by a track etching method (TE method). In the TE method, a porous film is formed by linearly damaging a film with radiation to form through-holes, and then widening the through-holes by chemical etching. Straight pores that penetrate from one side of the film to the other side are formed as through-holes in this porous film. The TE film thus formed is attached to the surface of the glass substrate 11 as a porous film. After this porous film is formed, a photothermal conversion region and a water-repellent film are formed on the porous film, similar to the above-described embodiment. This TE method can also be used to easily form a porous film with small pores.

[0064] 10(a) and 10(b) are tables showing the results of a demonstration experiment in which the generation of microbubbles MB in water was confirmed when water was poured onto a collection substrate manufactured according to the method for manufacturing a collection substrate for micro-objects in this embodiment.

[0065] Figure 10(a) shows the results of a demonstration experiment that confirmed the generation of microbubbles MB when the manufacturing method of the porous membrane, the average pore diameter [μm] of the pores in the porous membrane, the polymer material used in the porous membrane, the thickness [nm] of the material in the photothermal conversion region, and the silane coupling agent were each appropriately changed.

[0066] The generation status of microbubbles MB was confirmed by observing whether or not microbubbles MB were generated immediately after water was dispensed (dropped) onto the collection substrate using the sample supply device 90 and after the water was left to stand for 5 minutes. As shown in Fig. 2(b), this observation was performed by placing the porous membrane of the collection substrate near the focus of a laser beam irradiated from a laser light source 41, and observing whether or not microbubbles MB with a diameter of 100 μm or more were generated within 1 minute after the start of laser beam irradiation, immediately after water was dispensed onto the collection substrate and after the water was left to stand for 5 minutes. The drive current of the laser light source 41 was set to 180 mA, and the wavelength of the laser beam was set to 850 nm.

[0067] Comparative Example 1 and Example 1 shown in Figure 10(a) show the results of a demonstration experiment in which a porous film is formed by photolithography in the same manner as in the present embodiment described above, the average pore diameter of the pores in the porous film is 5 [μm], the material of the porous film is epoxy, the photothermal conversion material is Au, and the thickness of the collection substrate is 9 [nm], and in Comparative Example 1, the substrate surface was not treated with a silane coupling agent, while in Example 1, the substrate surface was treated with a silane coupling agent, n-octyltriethoxysilane.

[0068] In this demonstration experiment, in Comparative Example 1, where the substrate surface was not treated with a silane coupling agent, microbubbles MB were generated immediately after water was added, but no microbubbles MB were generated 5 minutes after water was added. On the other hand, in Example 1, where the substrate surface was treated with a silane coupling agent (n-octyltriethoxysilane), microbubbles MB were generated both immediately after water was added and 5 minutes after water was added, demonstrating the effectiveness of hydrophobic treatment of the substrate surface using a silane coupling agent. In other words, in Example 1, it was confirmed that air was continuously retained in the pores due to the hydrophobic treatment of the substrate surface using a silane coupling agent, and that microbubbles MB could be continuously generated using the air as a nucleus, even when laser light of the same intensity was irradiated onto the porous membrane in Comparative Example 1 and Example 1.

[0069] Comparative Example 2 and Example 2 show the results of a demonstration experiment in which a collection substrate has a porous membrane formed by the BF method, the pores of the porous membrane having an average pore diameter of 3.7 μm, the porous membrane material being polystyrene, the photothermal conversion material being Au, and the thickness being 10 nm.In Comparative Example 2, the substrate surface was not treated with a silane coupling agent, while in Example 2, the substrate surface was treated with the same silane coupling agent, n-octyltriethoxysilane, as in Example 1.

[0070] In this demonstration experiment, in Comparative Example 2, in which the substrate surface was not treated with a silane coupling agent, the generation of microbubbles MB was not confirmed both immediately after water was added and 5 minutes after water was added. On the other hand, in Example 2, in which the substrate surface was treated with the same silane coupling agent, n-octyltriethoxysilane, as in Example 1, the generation of microbubbles MB was confirmed both immediately after water was added and 5 minutes after water was added, as in Example 1, demonstrating the effectiveness of hydrophobic treatment of the substrate surface using a silane coupling agent.

[0071] Comparative Example 3 and Example 3 show the results of a demonstration experiment in which a porous film was formed by the BF method, the average pore size of the pores in the porous film was 2.5 [μm], which is smaller than that of Example 2, the material of the porous film was polystyrene, the photothermal conversion material was Au, and the thickness of the collection substrate was 10 [nm].In Comparative Example 2, the substrate surface was not treated with a silane coupling agent, while in Example 2, the substrate surface was treated with the same silane coupling agent, n-octyltriethoxysilane, as in Examples 1 and 2.

[0072] Comparative Example 4-1 and Example 4 show the results of a demonstration experiment in which a porous membrane was formed by the BF method, the average pore diameter of the pores in the porous membrane was 2.2 [μm], which is smaller than that of Example 2, the material of the porous membrane was polystyrene, the photothermal conversion material was Au, and the thickness of the collection substrate was 10 [nm].In Comparative Example 4-1, the substrate surface was not treated with a silane coupling agent, while in Example 4, the substrate surface was treated with the same silane coupling agent, n-octyltriethoxysilane, as in Examples 1 and 2.

[0073] In the demonstration experiments of Comparative Example 3 and Example 3, as well as Comparative Example 4-1 and Example 4, in Comparative Example 3 and Comparative Example 4, where the substrate surface was not treated with a silane coupling agent, no generation of microbubbles MB was confirmed both immediately after water was added and 5 minutes after water was added. On the other hand, in Examples 3 and 4, where the substrate surface was treated with the same silane coupling agent, n-octyltriethoxysilane, as in Examples 1 and 2, microbubbles MB were generated immediately after water was added, but no microbubbles MB were generated 5 minutes after water was added. These demonstration experiments showed that in Examples 3 and 4, where the average pore diameter was smaller than that of Example 2, no generation of microbubbles MB was confirmed both immediately after water was added and 5 minutes after water was added, as in Example 2, but microbubbles MB were generated immediately after water was added. This demonstrates that hydrophobic treatment of the substrate surface with a silane coupling agent improved the air retention performance of the pores.

[0074] Comparative Example 4-2 shows the results of a demonstration experiment carried out on a collection substrate in Example 4, where the average pore diameter of the pores in the porous membrane was set to 7.9 [μm], which is larger than that in Example 4, and the other conditions were the same as those in Example 4. This demonstration experiment demonstrated that when the average pore diameter of the pores in the porous membrane was set to 7.9 [μm], which is larger than 5.0 [μm], no generation of microbubbles MB was confirmed both immediately after water was added and 5 minutes after water was added, and it was demonstrated that the average pore diameter of the pores is preferably 5.0 [μm] or less.

[0075] Comparative Example 5 and Example 5-1 show the results of a demonstration experiment in which a porous membrane was formed by the TE method, the average pore diameter of the pores in the porous membrane was 5.0 [μm], the same as in Example 1, the material of the porous membrane was polyester, the photothermal conversion material was Au, and the thickness of the collection substrate was 10 [nm].In Comparative Example 5, the substrate surface was not treated with a silane coupling agent, while in Example 5-1, the substrate surface was treated with the silane coupling agent n-octyltriethoxysilane, the same as in Examples 1 to 4.

[0076] In this demonstration experiment, in Comparative Example 5, in which the substrate surface was not treated with a silane coupling agent, the generation of microbubbles MB was not confirmed both immediately after water was added and 5 minutes after water was added. On the other hand, in Example 5-1, in which the substrate surface was treated with the same silane coupling agent, n-octyltriethoxysilane, as in Examples 1 to 4, the generation of microbubbles MB was confirmed both immediately after water was added and 5 minutes after water was added, demonstrating the effectiveness of hydrophobic treatment of the substrate surface using a silane coupling agent.

[0077] Example 5-2 shows the results of a demonstration experiment conducted on a collection substrate in which the treatment with the silane coupling agent in Example 5-1 was performed with 3-aminopropyltrimethoxysilane instead of n-octyltriethoxysilane, and the other conditions were the same as in Example 5-1. This demonstration experiment confirmed that when the silane coupling agent was 3-aminopropyltrimethoxysilane, the generation of microbubbles MB was confirmed both immediately after water was added and 5 minutes after water was added, just as when n-octyltriethoxysilane was used, and demonstrated the effectiveness of hydrophobic treatment of the substrate surface with the silane coupling agent 3-aminopropyltrimethoxysilane.

[0078] Comparative Example 6 and Example 6 show the results of a demonstration experiment in which a collection substrate is formed with a porous membrane by the TE method, the pores of the porous membrane have an average pore diameter of 3.0 [μm], the material of the porous membrane is polyester, the photothermal conversion material is Au, and the thickness is 9 [nm].In Comparative Example 6, the substrate surface was not treated with a silane coupling agent, while in Example 6, the substrate surface was treated with an organoalkoxysilane silane coupling agent.

[0079] In this demonstration experiment, in Comparative Example 6, in which the substrate surface was not treated with a silane coupling agent, no generation of microbubbles MB was confirmed both immediately after water was added and 5 minutes after water was added. On the other hand, in Example 6, in which the substrate surface was treated with an organoalkoxysilane silane coupling agent, microbubbles MB were confirmed both immediately after water was added and 5 minutes after water was added, demonstrating the effectiveness of hydrophobic treatment of the substrate surface using an organoalkoxysilane silane coupling agent.

[0080] Comparative Example 7 and Example 7 show the results of demonstration experiments carried out on collection substrates in which the average pore diameter of the pores in the porous membrane in Comparative Example 6 and Example 6 was set to 5.0 [μm], respectively, and the other conditions were the same as those in Comparative Example 6 and Example 6.

[0081] In this demonstration experiment, in Comparative Example 7, in which the substrate surface was not treated with a silane coupling agent, the generation of microbubbles MB was not confirmed both immediately after water was added and 5 minutes after water was added. On the other hand, in Example 7, in which the average pore diameter of the pores of the porous membrane was set to 5.0 [μm], which is larger than that of Example 6, the generation of microbubbles MB was confirmed both immediately after water was added and 5 minutes after water was added, as in Example 6. Therefore, even when the average pore diameter of the pores in Example 6 was increased from 3.0 [μm] to 5.0 [μm], it was demonstrated that the effectiveness of the hydrophobic treatment of the substrate surface using the same organoalkoxysilane silane coupling agent as in Example 6 was confirmed.

[0082] Figure 10(b) shows the results of a demonstration experiment to confirm the generation of microbubbles MB in areas other than the laser light irradiation area for a collection substrate of a comparative example with pores formed on the entire surface of the substrate and a collection substrate of an example with pores formed only in the area irradiated with laser light. Both collection substrates are manufactured using the same photolithography method, the same average pore diameter of 5.0 μm, and the same silane coupling agent (n-octyltriethoxysilane), but the only difference is the pore distribution.

[0083] This demonstration experiment confirmed that, in the collection substrate of the comparative example, in which pores were provided over the entire surface of the substrate, microbubbles MB were generated in areas other than the laser beam irradiation area. On the other hand, in the collection substrate of the example, in which multiple pores were formed only in the area irradiated with laser beam, microbubbles MB were not generated in areas other than the laser beam irradiation area. Therefore, in the collection substrate of the comparative example, microbubbles MB generated in areas other than the laser beam irradiation area may become noise bubbles, which may cause the collected micro-objects α to disperse.

[0084] To summarize the above, the present invention can be expressed as follows.

[0085] <1> a porous film formed on the surface of the substrate and having a plurality of pores capable of retaining air; a photothermal conversion region formed on the surface of the substrate covering the porous film, the photothermal conversion region converting light irradiated from a light source into heat; a water-repellent film having water repellency formed on the surface of the substrate to cover the photothermal conversion region; a micro-object collection substrate for collecting a plurality of micro-objects dispersed in a liquid in which the porous membrane is immersed;

[0086] <2> The water-repellent film is formed by substituting hydrophilic groups on the surface of the photothermal conversion region with hydrophobic groups. <1> The minute object collection substrate according to claim 1.

[0087] <3> The pores have a vertical cross-sectional shape that is inversely tapered so that the opening area is smaller than the bottom area. <1> or <2> The minute object collection substrate according to claim 1.

[0088] <4> The pores have an opening diameter of 5 μm or less. <1> from <3> 10. The substrate for collecting minute objects according to any one of the preceding items.

[0089] <5> The porous film is characterized in that a plurality of pores are formed only in the area where light is irradiated from the light source. <1> from <4> 10. The substrate for collecting minute objects according to any one of the preceding items.

[0090] <6> forming a porous film having a plurality of pores capable of retaining air on a surface of a substrate; forming a photothermal conversion region that converts light irradiated from a light source into heat on the surface of the substrate that covers the porous film; and exposing the substrate surface to vapor of a silane coupling agent and water vapor for a predetermined time in an atmosphere at a predetermined temperature to form a water-repellent film on the substrate surface covering the photothermal conversion region. A method for producing a collection substrate for collecting a plurality of minute objects dispersed in a liquid in which the porous membrane is immersed.

[0091] <7> The porous membrane is formed by a breath figure method. <6> 10. A method for producing a substrate for collecting minute objects according to claim 1.

[0092] <8> The porous membrane is formed by a track etching method. <6> 10. A method for producing a substrate for collecting minute objects according to claim 1. [Explanation of symbols]

[0093] 1...Collection device 10...Collection substrate 11...Glass substrate 12...Resist film (pore film) 12a...pore 13...Thin film (photothermal conversion region) 14...Water-repellent film 15...Photomask 16...Air 17…liquid 18...Polymer solution 18a...Polymer membrane 19…Water droplet nucleus 19a...Water droplet 19b...pore 20...XYZ axis stage 30…Adjustment mechanism 41~49...Laser light source 50...Optical components 60...Objective lens 70...Lighting equipment 80...Filming equipment 90...Sample supply device 100...Control device S...Sample α…Minute object MB: Microbubbles LS1~LS9...Laser spot

Claims

1. a porous film formed on the surface of the substrate and having a plurality of pores capable of retaining air; a photothermal conversion region formed on the surface of the substrate covering the porous film, the photothermal conversion region converting light irradiated from a light source into heat; a water-repellent film having water repellency formed on the surface of the substrate to cover the photothermal conversion region; a micro-object collection substrate for collecting a plurality of micro-objects dispersed in a liquid in which the porous membrane is immersed;

2. 2. The micro-object collection substrate according to claim 1, wherein the water-repellent film is formed by substituting hydrophilic groups on the surface of the photothermal conversion region with hydrophobic groups.

3. 3. The minute object collection substrate according to claim 1, wherein the pores have a vertical cross section with an inversely tapered shape such that the opening area is smaller than the bottom area.

4. 3. The substrate for collecting minute objects according to claim 1, wherein the pores have an opening diameter of 5 [mu]m or less.

5. 3. The minute object collection substrate according to claim 1, wherein the porous film has a plurality of pores formed only in an area where light from a light source is irradiated.

6. forming a porous film having a plurality of pores capable of retaining air on a surface of a substrate; forming a photothermal conversion region that converts light irradiated from a light source into heat on the surface of the substrate that covers the porous film; and exposing the substrate surface to vapor of a silane coupling agent and water vapor for a predetermined time in an atmosphere at a predetermined temperature to form a water-repellent film on the substrate surface covering the photothermal conversion region. A method for manufacturing a micro-object collection substrate that collects a plurality of micro-objects dispersed in a liquid in which the porous membrane is immersed.

7. 7. The method for manufacturing a substrate for collecting minute objects according to claim 6, wherein the porous film is formed by a breath figure method.

8. 7. The method for manufacturing a micro-object collection substrate according to claim 6, wherein the porous film is formed by a track etching method.

Images