JP6679873B2 - Micro particle holder - Google Patents

Description

  The present invention relates to a device capable of holding microparticles from a liquid containing microparticles. In particular, the present invention relates to a device capable of reliably fractionating / sealing between holding units holding the fine particles.

  In recent years, cells have been selectively separated and collected from body fluids such as blood and tissues or tissue culture samples obtained by suspending or dispersing tissues such as organs in a solution. Research is being conducted to apply it to clinical diagnosis and treatment. For example, a tumor cell (Circulating Tumor Cell, hereinafter CTC) is collected from blood collected from a cancer patient, and morphological analysis, tissue type analysis, and gene analysis are performed on the cell, and based on the findings obtained by the analysis. Research to determine treatment policy is ongoing.

  Reference 1 uses a cell holding device provided with a holding portion capable of holding cells, and a cell holding device provided with a pair of electrodes for holding cells in the holding portion by a dielectrophoretic force. After the cells contained therein are efficiently retained in the holding part, the opening of the holding part is covered with a hydrophobic liquid such as mineral oil to fractionate / seal each holding part to achieve the sealing. It is disclosed that the gene analysis is carried out in the holding unit, and that the solution after the analysis is collected. However, in this method, the solutions contained in the holding part may communicate with each other via the surface of the cell holding means, and the resulting contamination may occur.

  As another method of fractionating / sealing the holding part, Patent Document 2 discloses that cells contained in a sample are held in the holding part by using dielectrophoretic force, and then a silicone resin film is applied to the holding part. A method of fractionating / sealing each holding part by joining the holding parts is disclosed. However, the method disclosed in Patent Document 2 has a problem that the solution cannot be collected from the sealed holding portion.

  As still another method of fractionating / sealing the holding portion, Patent Document 3 discloses a holding means provided with a holding portion having a hydrophobic upper surface and a side wall formed of amorphous fluororesin, in which each holding portion is provided. A method is disclosed in which, after accommodating the beads one by one, the respective holding parts are fractionated / sealed with a hydrophobic solvent such as a fluorine solvent. However, the method disclosed in the cited document 3 adopts a method in which the microparticles (cells) are settled by the gravity to the holding section as a method of holding the fine particles (cells) in the holding section. Therefore, in order to hold the microparticles (cells) not held in the holding part in the holding part, it is necessary to collect the solution containing the microparticles (cells) and re-introduce it into the holding means. There was a growing problem.

  As a method of covering the cell holding means with a hydrophobic film, in Patent Document 4, in a holding means provided with a holding portion capable of holding only one cell, the inner surface of the holding portion is covered with a fluorocarbon film or a silicon oxide film. By doing so, it is disclosed that the cells contained in the holding section can be easily recovered. However, the method disclosed in the cited document 4 also adopts a method in which the microparticles (cells) are settled to the holding part by gravity as a method of holding the microparticles (cells) in the holding part, similarly to the cited document 3, and the same problem as the cited document 3 Had a point. Further, according to the method disclosed in the cited document 4, when the inner surface of the holding portion is covered with a fluorocarbon film or a silicon oxide film, while the surface of the holding means has water repellency or hydrophobicity, the cells can be held in the holding portion. Since it becomes difficult, it is covered with a silicon oxide film having an intermediate property between hydrophilicity and hydrophobicity. Therefore, it is difficult to reliably fractionate / close each holding portion.

WO2011 / 149032 JP 2012-034641A WO2012 / 121310 WO2005 / 069001

  An object of the present invention is to provide a holding device provided with a substrate provided with a holding unit capable of holding fine particles, in which each holding unit holding the fine particles is reliably fractionated / sealed, and a solution in the holding unit is retained. It is to provide a device capable of collecting.

  In order to solve the above problems, the inventors of the present invention have earnestly studied, and as a result, arrived at the present invention.

  That is, a first aspect of the present invention is a fine particle holding device provided with a substrate provided with a holding portion capable of holding fine particles, wherein the surface of the substrate is treated with a water-repellent resin while the holding portion is provided. The inner surface of the device is treated so that a water-repellent resin film is not substantially formed.

  A second aspect of the present invention is the apparatus according to the first aspect, wherein the water repellent resin is a fluororesin.

Furthermore, a third aspect of the present invention is a method for analyzing microparticles, which includes the following steps (1) to (4) and uses the apparatus according to the first or second aspect.
(1) A step of introducing an aqueous solution containing fine particles into a substrate provided in the apparatus according to the first or second aspect (2) A step of holding the fine particles in a holding portion provided on the substrate (3) The above The step of sealing the holding part holding the fine particles with a hydrophobic solvent (4) the step of analyzing a specific substance present on the surface or inside of the fine particles held by the closed holding part. A fourth aspect is a method for recovering fine particles using the apparatus according to the first or second aspect, which includes the following steps (1) to (5).
(1) A step of introducing an aqueous solution containing fine particles into a substrate provided in the apparatus according to the first or second aspect (2) A step of holding the fine particles in a holding portion provided on the substrate (3) The above Step (4) of sealing the holding part holding the microparticles with a hydrophobic solvent, step (5) (4) of analyzing a specific substance existing on the surface or inside of the microparticles held by the closed holding part After the analysis of step 1), the step of recovering the fine particles or the aqueous solution containing the fine particles in the sealed holding portion. In a fifth aspect of the present invention, the step (2) is performed by utilizing dielectrophoretic force. The method according to the third or fourth aspect.

  Hereinafter, the present invention will be described in detail.

  Examples of the fine particles held in the holding unit by the holding device of the present invention include blood cells such as red blood cells and white blood cells, biological samples such as cancer cells and organ tissues, resin beads, ceramic powder, and metal fine particles. Note that the holding device of the present invention, after sealing the holding portion holding the fine particles, analyzes the specific substance existing on the surface or inside of the fine particles, and thus restricts the movement of the fine particles held by the holding portion. There is a need. As a limiting method, an appropriate method may be adopted depending on the properties of the fine particles. When the fine particles are magnetic, they may be held by applying a magnetic field to the holding portion (substrate). If the fine particles have an electric charge, they may be held by applying an electric field to the holding portion (substrate). If the fine particles are a dielectric substance such as cells, it is sufficient to apply an AC electric charge to the electrodes provided so as to sandwich the holding portion and hold the holding portion in the holding portion using the dielectrophoretic force. When the microparticle has a protein on its surface, a receptor for the protein or an antibody against the protein is immobilized on the holding part, and the protein-receptor interaction or antigen-antibody reaction is used to move to the holding part. Just install it. When the microparticles have a functional group on their surface, a ligand capable of specifically binding to the functional group may be immobilized on the holding part and introduced into the holding part by utilizing the specific binding. . In addition to these introductions, the movement of the fine particles held in the holding portion may be physically restricted by forming fine holes in the holding portion itself.

When the electric conductivity of the fine particles or the liquid to be dispersed is low (for example, a liquid containing cells or a liquid containing fine particles having an electric conductivity of 1 mS / cm or less (preferably 200 μS / cm or less)), dielectrophoretic force is used. It is preferable to introduce the fine particles into the holding portion because the fine particles can be efficiently introduced into the holding portion. When the dielectrophoretic force is used, specifically, dielectrophoresis is generated by applying an AC voltage, and the fine particles may be introduced into the holding portion. The AC voltage to be applied is preferably an AC voltage having a waveform in which charging / discharging of the microparticles in the holding part is periodically repeated. If the microparticles are cells, the frequency is between 100 kHz and 3 MHz, and the electric field strength is It is particularly preferable to set it in the range of 1 × 10 ⁵ to 5 × 10 ⁵ V / m (see WO 2011/149032 and JP 2012-013549 A).

  The fine particle holding device of the present invention is characterized in that the surface of the substrate provided with the holding portion capable of holding the fine particles is treated with a water-repellent resin. The water-repellent resin may be appropriately selected from water-repellent resins such as fluororesins and silicone resins. Among them, the fluororesin is preferable as the water-repellent resin for treating the surface of the substrate because it has high water repellency and high durability. Examples of the method of treating the surface of the substrate with the water-repellent resin include a method of directly applying the water-repellent resin to the substrate with a brush or the like, and a method of dropping the water-repellent resin onto the substrate and then spreading it over the entire substrate by spin coating. Note that the treatment of the substrate surface with the water-repellent resin may be performed so that the holding portion containing the aqueous solution containing fine particles can be sealed with a hydrophobic solvent so that the substrate surface is rendered hydrophobic. It is not necessary to form the film of the water-repellent resin, and the film of the water-repellent resin may be unevenly formed on the surface of the substrate. Further, if the treatment with the water-repellent resin is performed up to the inner surface of the holding portion, the water repellency of the inner space of the holding portion may be increased, which may make it difficult to hold the fine particles. The inner surface may be treated so that a water-repellent resin film is not substantially formed.

In the present specification, the term "water-repellent resin film is not substantially formed" means that the water-repellent resin film is formed in a region other than the region where the side surface of the holding portion contacts the substrate surface and the region where the side surface of the holding portion contacts the bottom surface. Is not formed. The area where the side surface of the holding portion contacts the substrate surface and the area where the side surface of the holding portion contacts the bottom surface may vary depending on the diameter and depth of the through hole of the holding portion and the material of the water-repellent resin used for the treatment. For example, when the diameter of the through hole (holding portion) is 30 to 50 μm, the depth of the through hole (holding portion) is 30 to 50 μm, and the water-repellent resin used for the treatment is a fluororesin,
The region where the side surface of the holding portion contacts the substrate surface is an area of 5% or less of the depth of the holding portion in the bottom surface direction of the holding portion from the tangent line between the side surface and the substrate surface on the side surface of the holding portion,
The area where the side surface and the bottom surface of the holding portion contact each other is 5% or less of the depth of the holding portion in the substrate surface direction from the tangent line between the side surface and the bottom surface on the side surface of the holding portion, and the bottom surface of the holding portion with the bottom surface. The area is 10% or less of the radius of the bottom surface and 20% or less of the bottom area of the holding portion in the center direction of the bottom surface from the tangent to the side surface.

  The microparticle holding device of the present invention can analyze a specific substance existing on the surface or inside of the microparticles by holding the microparticles in the holding part and then sealing the holding part with a hydrophobic solvent. it can. Further, after the analysis, the fine particles held in the holding unit or the aqueous solution containing the fine particles can be recovered. The hydrophobic solvent used to seal the holding part has no affinity for the solution (aqueous solution) held by the holding part, but has an affinity for the water-repellent resin used for the treatment of the substrate surface. Any solvent may be used, and examples thereof include oils such as mineral oil and fluorine solvents. As a method of sealing the holding portion, a sheet that can be in close contact with the surface of the substrate may be used instead of the hydrophobic solvent. An example of the sheet is a silicone rubber having optical transparency. The sealing method using the sheet is a useful sealing method for the purpose of analyzing a specific substance existing on the surface or inside of the microparticles, but the microparticles held in the holding part or an aqueous solution containing the microparticles. This is an unfavorable sealing method for the purpose of recovering. This is because, when the sealing by the sheet is released (the sheet is peeled from the substrate surface), the aqueous solution held by the holding portion may be evaporated or may be peeled off on the sheet side.

  The specific substance existing on the surface or inside of the microparticles is a substance existing on the surface or inside of the microparticles for specifying the target microparticles.When the microparticles are cells, proteins such as enzymes and , Genes such as DNA and RNA can be exemplified. As an example of the analysis method, there is a method of binding a specific substance and a fluorescent substance and directly observing fluorescence emitted from the fluorescent substance. The binding mode may be appropriately selected from covalent bond, electrostatic bond, antigen-antibody bond, ligand-receptor bond, biotin-avidin bond, hybridization between nucleotides, etc. in consideration of the mode of the specific substance. Good. When the specific substance is an enzyme, a method of observing fluorescence obtained by reacting the specific substance with a fluorescent substrate may be used. When the specific substance is a gene, a method of amplifying a nucleic acid containing a partial region of the gene (or simultaneously with the amplification) and then detecting the amplified nucleic acid by fluorescence or the like may be used. When recovering the microparticles or the aqueous solution containing the microparticles, a chip or a pipette may be directly inserted into the holding part to be recovered, or the hydrophobic solvent used for sealing may be removed before the recovery. You may perform the operation. Moreover, you may collect using the method as described in PLOS ONE, 10, e0130418 (2015). The present method is for a substrate provided with a holding portion capable of holding fine particles, the substrate having a holding portion having a through hole (holding portion) having a diameter of 30 μm and a through hole (holding portion) having a depth of 40 μm. This is a method in which cancer cells, which are held in the holding unit and are targets for collection, can be selectively collected by aspirating with a cell collection device equipped with a glass capillary having an inner diameter of 30 μm. According to this method, white blood cells that are not targets for collection held by adjacent holding units are not sucked at the same time.

An example of the fine particle holding device of the present invention is shown in FIG. The fine particle holding device 1 shown in FIG.
A plate-shaped insulator 11 having a through hole 11a,
A flat spacer 12,
Electrode substrates 21 and 22 provided so as to vertically sandwich the insulator 11 and the spacer 12 in close contact with each other;
A conductive wire 30 connecting the electrode substrates 21 and 22 to each other,
A signal generator 40 for applying a signal to the electrode substrates 21 and 22,
(Hereinafter, a mode in which the insulator 11 and the electrode substrate 21 are combined is referred to as a holding substrate 10). The electrode substrate 22 has an inlet 22a and an inlet 22b. The holding portion 50 is configured by the electrode substrate 21 provided in close contact with the through hole 11a and the lower portion of the insulator 11, and when a liquid containing cells is introduced from the introduction port 22a, fine particles are introduced into the holding portion 50 through the penetration portion 12a. To be done. The electrode substrate 22 is provided in close contact with the upper portion of the spacer 12 to prevent the liquid containing cells introduced from the inlet 22a from scattering and evaporating. Further, the electrode substrate 22 has a structure that can be removed from the spacer 12 in order to facilitate the recovery of the cells held in the holding unit 50.

  The present invention is a fine particle holding device provided with a substrate provided with a holding portion capable of holding fine particles, wherein the surface of the substrate is treated with a water-repellent resin while the inner surface of the holding portion is substantially It is characterized in that it is treated so that a film of water-repellent resin is not formed. In the device of the present invention, after holding the fine particles in the holding part, the holding part can be reliably fractionated / sealed by introducing the hydrophobic solvent. Therefore, in the subsequent analysis step (enzyme reaction, antigen-antibody reaction, nucleic acid amplification reaction, etc.), contamination between the holding parts can be prevented. Further, it is possible to collect the fine particles or the aqueous solution containing the fine particles from the holding unit that is fractionated / sealed.

The figure which showed an example of the microparticle holding | maintenance apparatus of this invention. (A) shows an exploded view and (B) shows a front view. The figure which showed each process of the cell analysis method using the microparticle holding | maintenance apparatus of this invention performed in Example 4 and the comparative example 1. The figure which showed the result of Example 4. (A) and (B) are a bright field image and a fluorescent image of the microparticle holding device of the present invention (holding portion diameter 30 μm) produced in Example 1, and (C) and (D) are in Example 2. 3A and 3B are a bright-field image and a fluorescent image of the produced microparticle holding device of the present invention (holding portion diameter 100 μm). The figure which showed the result of the comparative example 1. (A) and (B) are a bright field image and a fluorescent image of the fine particle holding device (holding portion diameter: 30 μm) produced in Comparative Example 1, and (C) and (D) are minute images produced in Comparative Example 1. 3 is a bright field image and a fluorescence image of a particle holding device (holding portion diameter 100 μm). FIG. 6 is a diagram showing each step of the cell analysis using the microparticle holding device of the present invention performed in Example 5 and the method of collecting target cells. The figure which showed the result of Example 5. (A) to (C) are bright field images, fluorescent images and cell nucleus staining images before collecting target cells, and (D) to (F) are bright field images, fluorescent images and cell nucleus staining images after collecting target cells. is there. FIG. 6 is a diagram showing each step of cell analysis using a method of sealing a holding portion with a silicone rubber sheet and a method of collecting target cells performed in Example 6.

  Hereinafter, the present invention will be described in more detail using examples and comparative examples, but the present invention is not limited to the examples.

Example 1 Preparation of microparticle holding device (holding portion diameter 30 μm)
The fine particle holding device 1 shown in FIG. 1 was produced by the method described below.
(1) A resist (epoxy type) is applied to the ITO film formation surface of the electrode substrate 21 (length 70 mm × width 38 mm × thickness 1 mm glass substrate on which ITO is formed) using a spin coater so as to have a film thickness of 30 μm. Negative resist).
(2) After natural drying for 1 minute, prebaking (95 ° C., 3 minutes) was performed using a hot plate.
(3) UV exposure using an exposure photomask in which a through hole pattern with a diameter of 30 μm is drawn, which is arranged in an array of 1000 in length × 600 in width in an area of 50 μm in length × 30 mm in width with an interval of 50 μm. The resist was exposed with a machine and developed with a developing solution. The exposure time and the development time were adjusted so that the depth of the through hole was 30 μm, which is equal to the film thickness of the resist, so that the ITO film formation surface of the electrode substrate 21 was exposed.
(4) The resist structure was solidified by post-baking (180 ° C., 30 minutes) using a hot plate. As a result, the holding substrate 10 provided with the holding portion 50, in which the insulator 11 having the through hole 11a was provided on the electrode substrate 21, was obtained.
(5) 1 mL of Fluorosurf FS-1000 series (manufactured by Fluorotechnology), which is a fluororesin coating agent, is dropped on the surface of the holding substrate 10 obtained in (4) on the side of the insulator 11 (the upper surface of the insulator 11) and the spin coater Was rotated at a rotation speed of 300 rpm for 5 seconds and subsequently at a rotation speed of 1500 rpm for 5 seconds to perform the treatment with the water-repellent resin.
(6) After the treatment with the water-repellent resin, baking was performed using a hot plate at 100 ° C. for 120 minutes to volatilize the volatile components. It was confirmed that the water contact angle of the surface of the insulator 11 after the treatment with the water-repellent resin was about 110 degrees, which was improved compared with the value before the treatment (less than 90 degrees). Energy dispersive X-ray analysis (EDS) mapping measurement was performed on the surface of the insulator film (upper surface of the insulator 11) after the treatment with the water-repellent resin. Although the resin film was formed in a patchy shape instead of the whole, there is no problem in carrying out the present invention. Further, as a result of performing the EDS mapping measurement on the inner surface of the holding portion after the treatment with the water-repellent resin in the same manner, the fluororesin film confirmed on the inner surface of the holding portion shows the substrate on the side surface of the holding portion from the tangent line between the side surface and the bottom surface. A region of 1 μm in the surface direction (about 3% of the depth of the holding portion) and 1.4 μm in the center direction of the bottom surface from the tangent line between the bottom surface and the side surface at the bottom surface of the holding portion (about 9% of the radius of the bottom surface. , About 18% of the bottom area of the holding portion). Therefore, it can be said that in this treatment method, the inside of the holding portion is treated so that the film of the water-repellent resin is not substantially formed.
(7) The spacer 12 was laminated on the upper part of the holding substrate 10 treated with the water-repellent resin and pressure-bonded. The spacer 12 is a flat plate made of silicon rubber having a thickness of 1.0 mm and provided with a through portion 12a having a length of 3.5 mm and a width of 10 mm. Since the surface of the spacer 12 has an adhesive property, each component can be brought into close contact with each other by pressure bonding. it can. The number of holding portions 50 (through holes 11a) existing in the through portion 12a is about 22,000.
(8) The electrode substrate 22 was laminated on the spacer 12 and pressure-bonded. The electrode substrate 22 is provided with an inlet 22a and an inlet 22b so that the liquid can be introduced / discharged to / from the holding portion 50 (through hole 11a) existing in the through portion 12a.
(9) By connecting the electrode substrates 21 and 22 and the signal generator 40 with a conducting wire 30, the fine particle holding device 1 shown in FIG. 1 was produced.

Example 2 Preparation of microparticle holding device (holding portion diameter 100 μm)
The fine particle holding device 1 shown in FIG. 1 was produced by the method described below.
(1) In the area of 50 mm in length × 30 mm in width of the electrode substrate 21 coated with the resist according to the method described in Example 1 (1) and (2), the array shape is 250 μm × 150 width × 200 μm. The resist was exposed by a UV exposure machine using the photomask for exposure in which a through-hole pattern having a diameter of 100 μm was arranged, and was developed with a developing solution. The exposure time and the development time were adjusted so that the depth of the through hole was 30 μm, which is equal to the film thickness of the resist, so that the ITO film formation surface of the electrode substrate 21 was exposed.
(2) The resist structure was solidified by post-baking (180 ° C., 30 minutes) using a hot plate. As a result, the holding substrate 10 provided with the holding portion 50, in which the insulator 11 having the through hole 11a was provided on the electrode substrate 21, was obtained.
(3) The treatment with the water-repellent resin was performed by the method described in Example 1 (5) and (6). By this treatment, the surface of the substrate (insulator 11) is treated with a water-repellent resin as in Example 1, but the inside of the holding portion is treated so that a film of the water-repellent resin is not substantially formed.
(4) The spacer 12 was laminated on the holding substrate 10 treated with the water-repellent resin and pressure-bonded thereto. The spacer 12 is a flat plate made of silicon rubber having a thickness of 1.0 mm and provided with a through portion 12a having a length of 3.5 mm and a width of 10 mm. Since the surface of the spacer 12 is adhesive, it is possible to bring the parts into close contact by crimping. it can. The number of holding parts 50 (through holes 11a) existing in the through part 12a is about 1375.
(5) The electrode substrate 22 was laminated on the spacer 12 and pressure-bonded. The electrode substrate 22 is provided with an inlet 22a and an inlet 22b so that the liquid can be introduced / discharged to / from the holding portion 50 (through hole 11a) existing in the through portion 12a.
(6) By connecting the electrode substrates 21 and 22 and the signal generator 40 with a lead wire 30, the fine particle holding device 1 shown in FIG. 1 was produced.

Example 3 Cell Retention Using Microparticle Retention Device Using the microparticle retention device 1 produced in Examples 1 and 2, cell retention was attempted by the method described below.
(1) Human metastatic pancreatic cancer cells AsPC-1 cells were cultured under RPMI-1640 medium containing 10% FBS, 2 mM glutamine and 1 mM pyruvic acid at 37 ° C. under 5% CO ₂ conditions. .
(2) After the cells reached subconfluence, they were washed with PBS (phosphate buffered saline), and trypsin-EDTA was used to remove the cells so that the individual cells were separated.
(3) The treated cells were centrifuged (200 xg at 25 ° C, 5 minutes), the supernatant was discarded, and then 0.1% bovine serum albumin (BSA) and 0.01 (v / v)% were added. The cells were suspended in a 300 mM mannitol aqueous solution containing Tween 20 to prepare a cell suspension having a cell density of 1 × 10 ⁵ cells / mL.
(4) 55 μL of the cell suspension prepared in (3) was introduced from the inlet 22a of the electrode substrate 22 using a syringe (the number of introduced cells: about 5,500), and the signal generator 40 generated a voltage of 20 Vpp and a frequency of 1 MHz. The rectangular wave AC voltage of was applied to the electrode substrates 21 and 22 for 3 minutes.

  As a result, it was confirmed that cells were held one by one in the holding unit 50 of the microparticle holding device 1 manufactured in Example 1. In addition, it was confirmed that a plurality of cells were held in the holding unit 50 of the microparticle holding device 1 manufactured in Example 2.

Example 4 Cell analysis using a microparticle holding device (with water-repellent resin treatment)
The cells retained in the retaining unit 50 by the method described in Example 3 were subjected to cell analysis by the method described below. The operation flow of this embodiment is shown in FIG.
(1) After holding the AsPC-1 cells 60 in the holding unit 50 by the method described in Example 3 (FIG. 2 (1)), the signal generator 40 continues to generate an alternating voltage (voltage 20 Vpp, rectangular wave of frequency 1 MHz). While applying 300mM mannitol aqueous solution containing 0.01 (w / v)% poly-L-lysine (adhesive substance 80) to the accommodating part while applying the electrode substrate 21/22, the application of AC voltage is stopped. Then, it was allowed to stand for 3 minutes (Fig. 2 (2)).
(2) Immediately after removing the solution in the microparticle holding device 1 by suction, introduce PBS into the cytodiagnostic chip from the introduction port 22a to wash the excess poly-L-lysine in the microparticle holding device 1. Then, the cells were electrostatically adhered into the micropores. Since the cells 60 are electrostatically adhered to the inside of the micropores by the action of poly-L-lysine, they are not detached from the holder 50 by the washing operation.
(3) After removing the solution in the microparticle holding device 1 by suction, PBS containing 10 μM ProteoGREEN ^™ -gGlu (manufactured by Goryo Chemical Co., Ltd.) was introduced from the inlet 22a into the accommodating portion, and allowed to stand at room temperature for 1 minute. ProteoGREEN ^™ -gGlu is a fluorescent imaging reagent 90 capable of selectively detecting cancer cells by being decomposed by an enzyme activity of cancer cells and emitting fluorescence (FIG. 2 (3)).
(4) After the solution in the fine particle holding device 1 was removed by suction, the holding part 50 was sealed by introducing the mineral oil 100 from the introduction port 22a (FIG. 2 (4)).
(5) By standing at room temperature for 60 minutes, the enzyme reaction between the enzyme possessed by the cancer cells and the fluorescent imaging reagent 90 proceeds, and the generated fluorescence 91 is detected by the optical detector 2 (Olympus inverted research microscope IX71). ) Through a CMOS camera (Fig. 2 (5)).

  The results are shown in FIG. From the bright field images (FIGS. 3 (A) and 3 (C)), it can be seen that the aqueous solutions in the holding parts of the microparticle holding device 1 produced in Examples 1 and 2 are fractionated from each other. In addition, from the fluorescence images (FIGS. 3B and 3D), the fluorescence derived from the cells was confirmed in the holding portion holding the cancer cells of the microparticle holding device 1 produced in Examples 1 and 2. 3 (B) and 3 (D), there is a difference in the fluorescence intensity in the holding part in which the cancer cells are held. However, due to the difference in the enzyme activity and the cell membrane state of the cancer cells held in the holding part. It is presumed to be the cause. From the above results, the surface of the substrate (insulator 11) is treated with the water-repellent resin, while the inner surface of the holding portion 50 is treated so that the film of the water-repellent resin is not substantially formed. It can be seen that the cells are fractionated from each other and the target cells can be reliably selected.

Comparative Example 1 Cell analysis using a microparticle holding device (without water-repellent resin treatment)
Among the microparticle holding devices 1 produced in Examples 1 and 2, those not treated with the water-repellent resin described in Examples 1 (5) and (6) were subjected to the same cell analysis as in Example 4. I tried.

  The results are shown in Fig. 4. From the bright-field images (FIGS. 4 (A) and 4 (C)), it is possible to confirm a plurality of locations where the liquid in the holding portion of the microparticle holding device 1 manufactured in Comparative Example 1 communicates with each other through the surface of the insulating film 11. It was Further, from the fluorescence images (FIGS. 3B and 3D), the fluorescence derived from the cancer cells held in the holding part of the microparticle holding device 1 produced in Comparative Example 1 is adjacent via the surface of the insulating film 11. It was confirmed that it was spreading to the holding part. From the above results, it can be seen that unless the surface of the substrate (insulator 11) is treated with a water-repellent resin, the fractionation of the aqueous solution in each holding part becomes insufficient, and it may be difficult to select the target cells. .

Example 5 Collection of target cells after cell analysis using microparticle holding device (with water-repellent resin treatment)
Using the microparticle holding device produced in Example 1, cells 60 held in holding unit 50 by the method described in Example 3 were analyzed by the method described below, and then target cells were tried to be collected. The operation flow of this embodiment is shown in FIG.
(1) After holding the cells 60 in the holding unit 50 by the method described in Example 3 (FIG. 5 (1)), the signal generator 40 continuously applies an alternating voltage (voltage 20 Vpp, rectangular wave of frequency 1 MHz) to the electrode substrate. After applying a 300 mM mannitol aqueous solution containing 0.01 (w / v)% poly-L-lysine (adhesive substance 80) to the accommodating portion while applying 21.22, the application of the AC voltage was stopped, and 3 It was allowed to stand for a minute (FIG. 5 (2)).
(2) Immediately after removing the solution in the microparticle holding device 1 by suction, introduce PBS into the cytodiagnostic chip from the introduction port 22a to wash the excess poly-L-lysine in the microparticle holding device 1. Then, the cells were electrostatically adhered into the micropores. Since the cells are electrostatically adhered to the inside of the fine pores by the action of poly-L-lysine, they are not detached from the holding part 50 by the washing operation.
(3) After suction-removing the solution in the microparticle holding device 1, a PBS containing 10 μM of ProteoGREEN ^™ -gGlu (manufactured by Goryo Kagaku) and 10 μg / mL of Hoechst 33342 (manufactured by Dojindo Laboratories) is accommodated from the inlet 22a. It was introduced into the chamber and left at room temperature for 1 minute. In addition, ProteoGREEN ^™ -gGlu is a fluorescent imaging reagent 90 capable of selectively detecting cancer cells by being decomposed by an enzyme activity of cancer cells and emitting fluorescence. Hoechst33342 is a reagent that stains the cell nucleus of living cells (FIG. 5 (3)).
(4) After the solution in the fine particle holding device 1 was removed by suction, the holding part 50 was sealed by introducing Fluorinert ^TM FC-40 (manufactured by 3M) (fluorine solvent 110) from the inlet 22a. Fluorinert ^™ FC-40 is an inert fluorine solvent (FIG. 5 (4)).
(5) By standing at room temperature for 60 minutes, the enzyme reaction between the enzyme possessed by the cancer cells and the fluorescent imaging reagent 90 proceeds, and the generated fluorescence 91 is detected by the optical detector 2 (Olympus inverted research microscope IX71). ) Through a CMOS camera (Fig. 5 (5)).
(6) The electrode substrate 22 of the microparticle holding device 1 was removed from the spacer 12, and target cells were collected by the cell collection method described in PLOS ONE, 10, e0130418 (2015) (FIG. 5 (6)).

  Results are shown in FIG. From the bright field image (FIG. 6 (A)), it can be seen that the aqueous solutions in the respective holding parts are fractionated from each other. Further, from the fluorescence image (FIG. 6 (B)), fluorescence derived from the cancer cells was confirmed in the holding portion holding the cancer cells. In FIG. 6 (B), although there is a difference in the fluorescence intensity in the holding part in which the cancer cells are held, it is presumed that it is caused by the difference in the enzyme activity and the cell membrane state of the cancer cells held in the holding part. It For example, when cancer cells having high enzyme activity (high fluorescence intensity) are used as target cells 61 and an attempt is made to perform suction collection using a cell collection device equipped with a glass capillary 120 having an inner diameter of 30 μm, the target cells 61 are collected by suction. We were able to. On the other hand, the non-target cells 62 held by the adjacent holding portions were not sucked by the cell collecting device (FIGS. 6D, 6E, and 6F). The target cells collected by suction could be visualized by a microscope by discharging them onto a slide glass. From the above results, the surface of the substrate (insulator 11) is treated with the water-repellent resin, while the inner surface of the holding portion 50 is treated so that the film of the water-repellent resin is not substantially formed. It is possible to prevent the contamination of the analysis results in the respective holding units between the respective holding units, and it is possible to reliably select the target cells. Furthermore, target cells selected from the analysis results in each holding part, which was impossible when the holding parts were fractionated / sealed by the method described in JP 2012-034641 A (Patent Document 2), The selective collection of is also possible in the present invention.

Example 6 Collection of target cells after cell analysis using microparticle holding device (holding holes sealed with silicone rubber sheet)
Using the microparticle holding device produced in Example 1, cells 60 held in holding unit 50 by the method described in Example 3 were analyzed by the method described below, and then target cells were tried to be collected. The operation flow of this embodiment is shown in FIG.
(1) After holding the cells 60 in the holding part 50 by the method described in Example 3 (FIG. 7 (1)), an AC voltage (voltage 20 Vpp, rectangular wave of frequency 1 MHz) is continuously applied by the signal generator 40 to the electrode substrate. After applying a 300 mM mannitol aqueous solution containing 0.01 (w / v)% poly-L-lysine (adhesive substance 80) to the accommodating portion while applying 21.22, the application of the AC voltage was stopped, and 3 It was allowed to stand for a minute (FIG. 7 (2)).
(2) Immediately after removing the solution in the microparticle holding device 1 by suction, introduce PBS into the cytodiagnostic chip from the introduction port 22a to wash the excess poly-L-lysine in the microparticle holding device 1. Then, the cells were electrostatically adhered into the micropores. Since the cells are electrostatically adhered to the inside of the fine pores by the action of poly-L-lysine, they are not detached from the holding part 50 by the washing operation.
(3) After removing the solution in the microparticle holding device 1 by suction, PBS containing 10 μM ProteoGREEN ^™ -gGlu (manufactured by Goryo Chemical Co., Ltd.) was introduced from the inlet 22a into the accommodating portion, and allowed to stand at room temperature for 1 minute. (FIG. 7 (3)).
(4) After the solution in the fine particle holding device 1 is removed by suction, the electrode substrate 22 and the spacer 12 are removed from the fine particle holding device 1 and immediately, a 1 mm thick silicone rubber sheet 130 made of polydimethylsiloxane (PDMS). Was pressed against the surface of the holding part 50 (FIG. 7 (4)).
(5) By standing at room temperature for 60 minutes, the enzyme reaction between the enzyme possessed by the cancer cells and the fluorescent imaging reagent 90 proceeds, and the generated fluorescence 91 is detected by the optical detector 2 (Olympus inverted research microscope IX71). ) Through a CMOS camera (FIG. 7 (5)).
(6) The silicone rubber sheet 130 is removed from the holding part 50, and the target cells 61, which are cancer cells with high enzyme activity (high fluorescence intensity), are collected by the cell collection method described in PLOS ONE, 10, e0130418 (2015). An attempt was made to collect (Fig. 7 (6)). However, the target cells 61 could not be collected because the liquid in the holding section 50 immediately evaporated.

1: Microparticle holding device 10: Holding substrate 11: Insulator 11a: Through hole 12: Spacer 12a: Through portion 21/22: Electrode substrate 22a: Inlet port 22b: Discharge port 30: Conductor 40: Signal generator 50: Hold Part 60: Cell 61: Target cell 62: Non-target cell 70: Dielectrophoretic force 80: Adhesive substance 90: Fluorescence imaging reagent 91: Fluorescence 100: Mineral oil 110: Fluorine solvent 120: Glass capillary 130: Silicone rubber sheet 2: Optical detector

Claims (5)

A fine particle holding device comprising a substrate provided with a holding portion capable of holding fine particles , which is formed by laminating an insulator having a through hole on an electrode substrate , wherein the insulator upper surface and the insulator upper surface are provided. It said apparatus characterized by said insulating body sides are in contact area and the insulator side face and the only water-repellent resin region and the upper surface of the electrode substrate is in contact with film is formed with. The device according to claim 1, wherein the water-repellent resin is a fluororesin. A method for analyzing microparticles, which uses the apparatus according to claim 1 or 2, which includes the following steps (1) to (4).
(1) A step of introducing an aqueous solution containing fine particles into a substrate provided in the apparatus according to claim 1 or 2 (2) A step of holding the fine particles in a holding portion provided on the substrate (3) The fine particles The step of sealing the retained holder with a hydrophobic solvent (4) the step of analyzing a specific substance present on the surface or inside of the microparticles retained by the sealed holder A method for recovering fine particles using the apparatus according to claim 1 or 2, which includes the following steps (1) to (5).
(1) A step of introducing an aqueous solution containing fine particles into a substrate provided in the apparatus according to claim 1 or 2 (2) A step of holding the fine particles in a holding portion provided on the substrate (3) The fine particles Step (4) of sealing the retained holding section with a hydrophobic solvent (4) Analyzing a specific substance present on the surface or inside of the microparticles retained by the sealed retaining section (5) (4) After that, the step of recovering the fine particles or the aqueous solution containing the fine particles in the sealed holding portion. The method according to claim 3 or 4, wherein the step (2) is performed by using dielectrophoretic force.

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