JP2008259491A - Flow channel substrate and reaction device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means which can simply extract cell contents such as nucleic acids even from a fine sample. <P>SOLUTION: A flow channel substrate 1 is formed by sequentially laminating filmy electrodes and a photosensitive resin layer 12 on a glass support 11, and the photosensitive resin layer 12 is partially removed to form solution tanks 21, 21 and a flow channel 23. A cell-crushing portion 6 and a filter 4 are sequentially disposed in the halfway of the flow channel 23. Thereby, when a driving voltage is applied to driving electrodes 51 formed on the bottom surfaces of the solution tanks 21, 21, a cell C in a sample solution charged in the flow channel 23 is moved. The cell C is nipped in a cell-catching portion 61 having narrowed flow channel widths, and crushed with the high frequency voltage between crushing electrodes 62, 62. The inner contents C1 of the cell C are flowed out in the flow channel 23. The cell membranes C2 and the like among the contents are hooked on the filter 4 disposed in the downstream, and nucleic acid and the like are flowed to the other solution tank 21. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a flow path substrate in which fine flow paths are formed, and a reaction apparatus including the flow path substrate.

  Conventionally, in order to extract nucleic acids from cells, for example, a nucleic acid extraction kit as shown in Patent Document 1 has been used. By using this, nucleic acid can be extracted from the cells, but the operator has to dispense a small amount of various reagents according to the manual, and the operation is complicated and requires time. Further, when the operation is performed in multiple stages, there is a possibility that impurities may be mixed in the process of operation, and there is a problem that nucleic acid extraction from a very small sample is difficult because yield occurs at each stage.

On the other hand, Patent Document 1 discloses a method of using silica particles as a nucleic acid-binding solid phase carrier. According to this method, nucleic acids can be extracted in one step, so that the operation is simpler and quicker. It can be performed.
JP-A-11-169170

  However, the method of Patent Document 1 has a problem that the yield of nucleic acid becomes insufficient because substances other than nucleic acids of cells inhibit the adsorption of nucleic acids to silica particles. Have difficulty.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide means capable of easily extracting cell inclusions such as nucleic acids from a minute sample.

  In addition, since the flow path is fine and extends along the surface, it was introduced into the flow path depending on environmental conditions, for example, when illuminated by a microscope for a long time, or when it was hot or dry. In some cases, the liquid evaporates and falls below the required amount.

  The present invention has been made in view of the above-described problems, and an object thereof is to prevent evaporation and drying of liquid in the flow path of the flow path substrate.

  In order to solve the above problems, a flow path substrate according to claim 1 of the present invention is formed so that a flow path extends along a surface, and cells in a solution introduced into the flow path are placed in the flow path. In the flow path substrate to be moved, a filter is provided in the middle of the flow path to separate the inclusions and dust of cells moving in the flow path according to the size. Here, the size means not only mass but also three-dimensional size.

  Thus, by providing a size filter in the middle of the flow path, inclusions of a size other than the target can be separated and removed, and the target inclusion can be purified.

  The flow path substrate according to claim 2 of the present invention includes a cell crushing unit that crushes cells contained in the solution upstream of the filter according to claim 2, and the filter is formed by the cell crushing unit. The inclusions in the solution that moves in the channel after cell disruption are separated according to their sizes.

  In this way, combining the cell disruption unit and the size filter makes it easy and quick to perform a series of operations from removing impurities, especially large inclusions after cell disruption, to recovering nucleic acids and target cell inclusions. Can be done. In addition, by performing a series of flows in this way, it is possible to suppress the contamination of impurities accompanying the replacement of the container and pipetting operation, and also to suppress the loss of the sample. It can be carried out. In addition, the extraction part for performing extraction in steps downstream can also be provided in the downstream of a filter, In this case, the extraction efficiency in a downstream extraction process can be improved by using a size filter.

  The flow path substrate according to claim 3 of the present invention is the flow path substrate according to claim 1 or 2, wherein the filter prevents passage of substances other than inclusions to be separated and extracted. Features.

  According to a fourth aspect of the present invention, in the flow path substrate according to any one of the first to third aspects, the filter includes at least one structure that divides the cross section of the flow path into a plurality of sections. And

  The flow path substrate according to claim 5 of the present invention is characterized in that, in claim 4, the structure is a columnar member extending in an out-of-plane direction from a bottom surface of the flow path.

  The flow path substrate according to claim 6 of the present invention is characterized in that, in claim 5, the filter is composed of a plurality of structures arranged side by side in the flow path width direction.

  The flow path substrate according to claim 7 of the present invention is the flow path substrate according to claim 5 or 6, wherein the structure has a shape in which a portion facing the upstream side of the flow path projects from the downstream side toward the upstream side. Features.

  A flow path substrate according to an eighth aspect of the present invention is the flow path substrate according to any one of the first to seventh aspects, wherein a pair of driving electrodes for moving the sample molecules in the flow path is located upstream of the flow path. And are integrally formed on the downstream side.

  The flow path substrate according to claim 9 of the present invention is the flow path substrate according to any one of claims 1 to 8, wherein the flow path is formed by partially laminating a photosensitive resin layer on a plate-like support. It is characterized by.

  According to a tenth aspect of the present invention, in any one of the first to ninth aspects, the flow path substrate includes a cooling unit that cools the flow path substrate. According to this, since the flow path substrate is cooled, evaporation of the liquid in the flow path is suppressed, and a long-time operation or the like is possible.

  The cooling part is preferably a Peltier element. Moreover, it is preferable that the Peltier element is disposed so that the endothermic surface thereof is in contact with the bottom surface of the flow path substrate. Moreover, it is preferable to provide the 2nd cooling part which cools the thermal radiation surface of the said Peltier element.

  A flow path substrate according to a fourteenth aspect of the present invention is characterized in that, in any one of the first to thirteenth aspects, a micropump is provided to move the cells in the flow path.

  The micropump includes a magnetic object and a non-magnetic object movably disposed in a pump flow path communicating with the flow path, and a plurality of electromagnets disposed along the pump flow path. By supplying electric power to an electromagnet close to the magnetic object, the magnetic object is moved together with the non-magnetic object in a predetermined direction in the pump flow path by the generated magnetic force. The cell in the solution is moved in the flow path.

  According to the micropump, by supplying electric power to the electromagnet close to the magnetic object, the magnetic object is moved together with the nonmagnetic object in a predetermined direction in the pump flow path by the generated magnetic force. Therefore, the fluid can be guided in a certain direction, so that the fluid can be pumped without using a motor, a diaphragm, etc., and it has a very simple structure, can be easily downsized, and has high durability. With such a micropump, cells can flow in the channel together with the solution in the channel substrate.

  A reaction apparatus according to a sixteenth aspect of the present invention is a reaction apparatus including the flow path substrate according to any one of the first to thirteenth aspects, and further includes a driving unit that moves the cells in the flow path. It is characterized by.

  A reaction apparatus according to claim 17 of the present invention is a reaction apparatus comprising the flow path substrate according to claim 14 or 15, wherein the cells are moved in the flow path by the micropump.

  According to each of the above-described reaction apparatuses, a reaction that can separate and remove inclusions of an undesired size with a filter provided in the middle of the flow path while moving cells in the flow path, and purify the desired inclusions. A device can be realized.

  A reaction apparatus according to an eighteenth aspect of the present invention is the reaction apparatus according to the sixteenth or seventeenth aspect, further comprising a cell crushing apparatus that crushes the cells contained in the solution on the upstream side of the filter. A cell capturing unit that is provided on the flow path substrate and captures the cells on the upstream side of the filter; and a crushing voltage applying unit that applies a voltage capable of crushing the cells to the cells captured by the cell capturing unit; It is characterized by comprising. Thereby, the reaction apparatus which can crush a cell upstream from a filter, isolate | separate and remove the inclusion of the size other than the target with a filter, and refine | purify the target inclusion can be implement | achieved.

  A flow path substrate according to a nineteenth aspect of the present invention is used in any one of the first to thirteenth aspects, attached to a centrifuge device to move a solution or cells in the solution in the flow path of the flow path substrate. It is characterized by being. According to this, the solution or cells in the solution can be moved in the flow path of the flow path substrate by attaching the flow path substrate to the centrifuge and rotating it to apply centrifugal force. For this reason, it is not necessary to apply an electric field for generating an electroosmotic flow, so that there is no influence on the substance to be detected by the application of the electric field.

  The centrifuge includes a rotating disk that attaches the flow path substrate containing the solution and rotates around a predetermined rotation axis, and a microscope for visually observing the state of cells in the solution in the flow path substrate. And a visible centrifuge that separates or synthesizes predetermined cells from the solution by applying a centrifugal force to the solution in the channel substrate or the cells in the solution.

  According to the present invention, the target substance can be purified simply and quickly by providing the size filter in the middle of the flow path.

  Next, embodiments of the present invention will be described with reference to the drawings.

  <First Embodiment>

  FIG. 1A is a plan view of a flow path substrate according to the present embodiment, FIG. 1B is a diagram showing a stacked configuration of the flow path substrate of FIG. 1A, and FIG. FIG. 3 is a cross-sectional view taken along line I, and FIG. 3 is a diagram for explaining the effect of the present embodiment ((a) shows a state in which cells are crushed, and (b) shows a state in which inclusions are separated after crushing).

(About flow path substrate)
As shown in FIG. 1B, the flow path substrate 1 is formed by sequentially laminating a film-like electrode and a photosensitive resin layer 12 on a glass support 11. The support 11 is a plate-like support that supports the membrane electrode and the photosensitive resin layer 12. In the present embodiment, a commercially available plate-like borosilicate glass (trade name: Pyrex (registered trademark)) having a length of 20 mm, a width of 10 mm, and a thickness of 1 mm is used as the support 11.

  The film-like electrodes are formed in a predetermined pattern on the support 11, thereby constituting a pair of driving electrodes 51 and 51 and a pair of crushing electrodes 62 and 62 described later. The respective electrodes 51, 51, 62, 62 are formed so as to be separated from each other and extend to the end portion of the support 11 so as to be connectable to the external electrode at the end portion. In FIG. 1 (a), the formation region of the film electrode is indicated by hatching for easy understanding. Among these, the hatched portion of the broken line indicates a portion formed under the photosensitive resin layer 12, and the hatched portion of the solid line indicates a portion where the film-like electrode is exposed. In this embodiment, after a mask pattern is formed on the support 11 using a resist or the like, a film electrode having a two-layer structure is formed by sputtering and covering the surface with a Pt reaction protective film (see FIG. 1 (b)). Thus, the adhesion with the support 11 can be improved by using Ti as a base, and the electrode reaction when exposed to the sample solution can be suppressed by covering with Pt.

  The photosensitive resin layer 12 is formed by photoreacting a photosensitive resin, and a predetermined two-dimensional pattern is formed on the surface of the support 11 with a predetermined thickness. Due to the difference in thickness between the formed portion of the photosensitive resin layer 12 and the unformed portion, a recess (described later) 2 for storing the sample solution is formed on the surface of the flow path substrate 1. In this embodiment, a negative photoresist (trade name: SU-8) manufactured by Kayaku Microchem Co., Ltd. is applied to the support 11 with a spin coater, exposed to ultraviolet light through a pattern mask, and undeveloped with a developer. The photosensitive resin layer 12 having a thickness of 25 μm is formed by dissolving and removing the cured portion.

  Next, the configuration of the recess 2 will be described.

  In FIG. 1A, the recess 2 is composed of a channel 23 extending in the lateral direction and circular solution tanks 21 and 21 connected to both ends of the channel 23 in the channel direction, respectively. . In the present embodiment, the cell crushing part 6 and the filter 4 are installed in the middle of the flow path 23.

  The cell crushing unit (or cell crushing device) 6 includes a cell trapping unit 61 and a pair of crushing electrodes 62 and 62. The cell trapping portion 61 is a portion of the flow path 23 that is formed so that the flow path width becomes narrower from the upstream side to the downstream side (in the drawing, from the left side to the right side in the drawing) in the flow path direction. The narrowest part of the cell trapping part 61 is set smaller than the diameter of the cell to be crushed. Further, the pair of crushing electrodes 62, 62 are formed in the vicinity of both ends in the flow path direction of the cell trapping part 61 with a space therebetween, and receives a power supply from an external power source to receive a high frequency for cell crushing. A voltage (for example, 0.1 to 1 MHz) is applied.

  In addition, the filter 4 includes a pair of structures 45 and 45 and is provided immediately downstream of the cell trap 61. The structure 45 is a columnar member that extends from the bottom surface of the flow path 23 and has a diameter smaller than the width of the flow path (see FIG. 2), and is formed of the photosensitive resin layer 12 together with the side wall of the flow path 23 and the like. In the flow path 23, a pair is arranged along the width direction of the flow path 23, and between the structures 45, 45 and between the structures 45, 45 and the side wall of the flow path 23. A slight gap is formed.

  In the present embodiment, the flow path 23 has a width of 20 μm on the left side of the paper with the cell trapping portion 61 interposed therebetween, and the narrowest portion of the cell trapping portion 61 is 10 μm. The width is set slightly wider than the left portion of the paper, and the depth is 25 μm.

  The solution tanks 21 and 21 are used for charging or collecting the sample solution. A pair of drive electrodes 51, 51 are formed in the formation regions of the solution tanks 21, 21, respectively, and are exposed on the surface, and constitute the bottom surfaces of the solution tanks 21, 21. The pair of driving electrodes 51 and 51 receives a power supply from an external power source and applies a driving voltage for forming an electric field for driving the sample solution. This drive voltage may be a DC voltage (for example, 30 V) or an AC voltage.

(About the effect of the channel substrate)
Next, the effect of the flow path substrate 1 having the above configuration will be described with reference to a usage example shown in FIG.

  When the flow path substrate 1 is used, each electrode of the flow path substrate 1 is connected to an electrode of an external power source so as to be operable. Then, a sample solution containing cells C as sample molecules is put into the solution tank 21 of the flow path substrate 1. In this state, the external power supply is turned on, and a drive voltage is applied to the pair of drive electrodes 51 and 51. In this use example, an electroosmotic flow from the positive electrode side toward the negative electrode side is generated by applying a DC voltage with the driving electrode 51 on the left side of the paper as the solution tank 21 side as a positive electrode and the other as a negative electrode.

  When the electroosmotic flow is generated, the cell C in the solution also moves in the channel 23 from the positive electrode side toward the negative electrode side. Since the width of the channel 23 becomes narrower from the upstream side toward the downstream side in the cell trapping part 61 of the cell crushing unit 6, the cell C flowing through the channel 23 is narrower than its diameter. The portion is sandwiched between the side walls and stopped (see FIG. 3A). In this state, when a high frequency voltage is applied to the crushing electrodes 62, 62, the cells C are energized and crushed. As a result, the inclusion (nucleic acid, protein, etc.) C1 elutes into the sample solution, and the cell membrane C2 and the like after releasing the inclusion C1 together with the electroosmotic flow again toward the solution tank 21 on the negative electrode side. Start moving. At this time, since the structures 45 are provided immediately downstream of the cell crushing portion 6, a substance having a relatively large size such as the cell membrane C <b> 2 is formed on the curved surface protruding toward the upstream side of the structure 45. Collide and get caught around the structure 45. Alternatively, the gap formed between the structures 45 and 45 or between the structure 45 and the side wall cannot be easily slipped through, and is stopped as it is or the moving speed is decreased. On the other hand, even if a relatively small substance such as a nucleic acid collides with the structure 45, the substance moves between the structures 45, 45 or between the structure 45 and the side wall. Therefore, only the relatively small inclusion C1 reaches the negative electrode side (right side of the drawing in the figure), or the inclusion C1 reaches the solution tank 21 earlier, so that the cell membrane C2 And the inclusion C1 alone can be recovered.

(Other configuration examples)
The application of the present invention is not limited to the above embodiment. For example, in the above embodiment, the structures 45, 45 are configured as columnar members extending from the bottom surface of the flow path. However, the structures 45, 45 are not limited to the columnar shape, and may be configured by a triangular columnar member as shown in FIG. . At this time, if one apex is arranged so as to face the upstream side, and the portion facing the upstream side is projected toward the upstream side, a large-sized substance such as a cell membrane is hooked so as to be wound, and from other molecules Can be separated.

  Note that the structure 45 is not limited to the case where the portion facing the upstream side protrudes toward the upstream side, and may be a concave shape that receives the flowing material. By adopting a concave shape, a substance having a small size can be retained in the recess, and separated from a substance having a large size that continues to flow without staying in the recess.

  Moreover, in the said embodiment, although the structures 45 and 45 are arrange | positioned in 1 row along the width direction of the flow path 23, you may arrange | position 2 or more rows. In this case, adjacent rows of structures 45 may be arranged in a staggered manner so that the molecular flow paths formed by the gaps between these structures 45 bend. Further, the arrangement is not limited to the case where a plurality of lines are arranged along the width direction, and the lines may be arranged in a line along the flow path direction. In this case, instead of arranging them in a straight line along the flow path direction, for example, one structure is closer to one side in the flow path width direction, and the next structure arranged on the downstream side is opposite to the flow path width direction. By alternately arranging on the side, the flow path of molecules formed by the gaps between them may be bent labyrinth.

  1 to 4, the structure 45 is disposed away from the side wall of the flow path 23, but may be disposed so as to be in contact with the side wall or may be integrally formed with the side wall.

  In addition, the driving method of the cells in the solution is not limited to the case of the electroosmosis phenomenon as in the above embodiment, and may be a method of driving with a pressure flow generated by a pressure difference such as when using a pump. Alternatively, a micro pump as described below may be used.

  The driving electrodes 51 and 51 and the crushing electrodes 62 and 62 are not limited to being integrally formed on the support 11 as film-like electrodes. For example, the driving electrodes 51 and 51 and the crushing electrodes 62 and 62 are configured as wire electrodes and inserted into the flow path 23. A voltage may be applied.

  Moreover, the constituent material of the flow path substrate 1 is not limited to that shown in the above embodiment. For example, the support 11 may be made of any material having a necessary strength as a support. However, in addition to glass (borosilicate glass, quartz glass, etc.), for example, plastic (polystyrene, polymethyl methacrylate). , Polysulfone, polyester, etc.) and glass fiber and plastic composites can also be used.

  The film electrode may be a general electrode material as well as the above materials, but the surface portion has a relatively high standard electrode potential (such as a positive value) such as Pt, Au, or Ag. ) It is preferable to use a material because it can prevent electrolytic corrosion when exposed to the sample solution. In addition, when a transparent electrode such as ITO (Indium Tin Oxide) is used, the transparency of the flow path substrate 1 can be maintained, which is preferable when optical analysis of the flow path substrate 1 is performed. Moreover, although it can improve adhesiveness with the support body 11 by forming by sputtering, it can also form by not only this but chemical vapor deposition, ion plating, and other physical vapor deposition.

  The photosensitive resin is not limited to the negative photosensitive resin that cures the portion irradiated with light as shown in the above embodiment, and a positive photosensitive resin that dissolves the portion irradiated with light is also used. Can do. However, from the viewpoint of securing the thickness and strength of the layer, a negative photosensitive resin that becomes a cross-linked polymer by a polymerization reaction during photocuring is preferable. The polymerization reaction may be any of radical polymerization, anionic polymerization, cationic polymerization and the like. As the photosensitive resin forming the crosslinked polymer, a known photosensitive resin containing a monomer and / or oligomer as a main component and further containing a photopolymerization initiator and various additives (stabilizer, filler, pigment, etc.) should be used. Can do. As this monomer, for example, (meth) acrylic monomers such as diethylene glycol di (meth) acrylate and trimethylolpropane tri (meth) acrylate can be used. Moreover, as an oligomer, the (meth) acrylic acid ester of an epoxy resin, the (meth) acrylic acid ester of a polyether resin, and the polyurethane resin which has a (meth) acryloyl group in a molecule terminal can be used, for example. As the photopolymerization initiator, for example, a benzoin photopolymerization initiator (benzoin, benzoin methyl ether, etc.) or an acetophenone photopolymerization initiator (2-2′-diethoxyacetophenone, etc.) can be used.

  Further, the arrangement of the flow paths 23 and the solution tanks 21 and 21 is not limited to that shown in FIG. 1. For example, a plurality of flow paths 23 are arranged side by side or other functional units (for example, extraction units) are provided. It may be provided on the downstream side of the filter 4.

  <Second Embodiment>

  In the second embodiment, the above-mentioned flow path substrate is provided with a cooling unit such as a Peltier element in a reaction apparatus.

  FIG. 5 is a functional block diagram of the reactor according to the second embodiment, FIG. 6 is a diagram showing the appearance of the reactor ((a) is a front view, (b) is a plan view, and (c) is a side view). FIG. 7 is a diagram showing a schematic configuration of the Peltier element used in the present embodiment.

  In the reaction apparatus of the present embodiment, as shown in FIGS. 6A to 6C, a support plate 33b is horizontally attached to an L-shaped base 50, and the upper surface side of the support plate 33b. A flat Peltier element 31 is fixed to the upper surface of the Peltier element 31, and the flow path substrate 1 is placed so as to be in close contact with the upper surface of the Peltier element 31. A connector 41 is attached on the support plate 33b, and the flow path substrate 1 is inserted into a connection port of the connector 41. The connector 41 can be connected to the voltage application device 42 shown in FIG. 5 through the connection port 41 b on the opposite side, and electrically connects the flow path substrate 1 and the voltage application device 42. A water cooler body 33a is attached below the support plate 33b.

  The flow path substrate 1 is formed with a flow path 23 along a plane, and is configured so that the sample solution introduced into the flow path 23 can flow by applying a voltage. The configuration is the same as in FIG.

  The Peltier element 31 is a thermoelectric conversion element that absorbs or dissipates heat by the Peltier effect, and has a configuration as shown in FIG. 7 in the present embodiment, and either the front surface or the back surface serves as a heat absorption surface, and the other surface dissipates heat. A Peltier element is used as the surface. FIG. 7 shows a part of the Peltier element 31. The Peltier element 31 includes a plurality of P-type thermoelectric semiconductors 31p and N-type thermoelectric semiconductors 31n arranged between a pair of substrates 31a and 31a, and these are arranged on the substrate 31a. The stacked copper electrodes 31c are alternately connected in series. As shown in FIG. 7, when a current is passed from the N-type thermoelectric semiconductor 31n toward the P-type thermoelectric semiconductor 31p, the surface facing the upper side of the paper becomes a heat absorbing surface, and the surface facing the lower side of the paper becomes a heat radiating surface. On the other hand, when a current flows in the opposite direction, the surface facing the upper side of the paper becomes a heat radiating surface, and the surface facing the lower side of the paper becomes a heat absorbing surface. In the reactor, as described above, the Peltier element 31 is brought into close contact with the flow path substrate 1 to adjust the temperature of the flow path substrate 1.

  Further, a temperature sensor 34 is attached to the upper surface side of the Peltier element 31 as shown in FIG. 6B so that the temperature of the contact surface with the flow path substrate 1 can be detected. The temperature sensor 34 uses, for example, a thermistor whose resistance changes according to temperature. In addition, the kind of temperature sensor to be used is not limited to this.

  Further, the Peltier element 31 is electrically connected to a Peltier controller 32 shown in FIG. 5 through a pair of conductive wires 31b and 31b. The drive current from the Peltier controller 32 is received and adjusted to a target temperature. The As shown in FIG. 5, the Peltier controller 32 receives the detection signal of the temperature sensor 34, and based on this, calculates the magnitude of the drive current for bringing the flow path substrate 1 to the target temperature. The drive current adjusted to the magnitude is output. The Peltier element 31 corresponds to a cooling unit.

  As shown in FIGS. 6A to 6C, the water cooler 33 includes a metal support plate 33b and a water cooler body 33a attached to the lower surface side of the support plate 33b. The water cooler body 33a is provided with a coolant passage in the metal block, and the coolant introduced from the inlet 33c is discharged from the outlet 33d through the coolant passage. Yes. This cooling liquid may be drawn directly from the water supply, or a secondary cooling device provided with a pump or a radiator may be provided to circulate the cooling liquid cooled by the secondary cooling device. The water cooler 33 corresponds to a second cooling unit.

  When the reaction apparatus having the above configuration is used, the flow path substrate 1 is attached to the connector 41, the voltage application device 42 is turned on, and a drive voltage is applied to the drive electrodes 51, 51 of the flow path substrate 1. Then, the sample solution introduced into the recess 2 is driven. At the same time, the Peltier controller 32 is also turned on. When the Peltier controller 32 detects that the endothermic surface of the Peltier element 31 exceeds the preset temperature, the Peltier element 31 is driven to cool the flow path substrate 1. As a result, the flow path substrate 1 is kept at a preset temperature or lower, so that evaporation of the sample solution is suppressed. For this reason, when the work requires a long time, the sample solution does not dry up during the work, and the work can be performed while maintaining a certain amount of solution. In this case, the cells can be crushed by applying a high-frequency voltage to the crushing electrodes 62, 62 of the flow path substrate 1 from an external power source (not shown).

  Note that the present invention is not limited to the second embodiment. For example, the means for cooling the flow path substrate 1 is not limited to the Peltier element 31, and for example, a heat sink is disposed so as to contact the flow path substrate, and the fan is blown toward the heat sink to cool it. May be. However, there is a risk that the sample solution may be easily dried unless air is applied to the flow path substrate 1 during air cooling. In this respect, it is preferable to use the Peltier element 31. Further, when the Peltier element 31 is used, high-precision cooling is possible, so that it is possible to maintain a limited temperature range even when handling a biopolymer having a high temperature sensitivity, and the apparatus can be downsized and quiet. In addition, it is preferable because it can operate without vibration.

  6 (a) to 6 (c) have a configuration in which only one flow path substrate 1 is mounted, a configuration in which a plurality of connectors 41 are provided and a plurality of flow path substrates 1 can be mounted simultaneously. It is good. In this case, a plurality of Peltier elements 31 may be provided and cooled for each flow path substrate 1, or a plurality of flow path substrates 1 may be mounted on a single Peltier element 31. If a plurality of Peltier elements 31 are provided and can be controlled for each Peltier element 31, each flow path substrate 1 can be cooled with high accuracy.

  Further, in order to enhance the adhesion between the Peltier element 31 and the flow path substrate 1, a mechanism for pressing the flow path substrate 1 downward may be provided in the connector 41, or silicon grease may be applied.

  Further, the flow path substrate 1 is not limited to the above configuration. For example, the number of the flow paths 23 may be two or more. In the second embodiment, the temperature sensor 34 is provided on the Peltier element 31 side. However, the temperature sensor 34 may be provided on the lower surface of the flow path substrate 1 or may be formed integrally with the recess 2 of the flow path substrate 1. Good. Further, the flow path substrate 1 and the Peltier element 31 may be integrally formed on one chip.

  <Third Embodiment>

  The third embodiment is a flow path substrate in which a solution is made to flow using a micropump that pumps a small amount of liquid.

  FIG. 8 is a side cross-sectional view of the micropump according to the third embodiment. FIG. 9 is a diagram of the configuration of FIG. 8 taken along the line II-II and viewed in the direction of the arrow.

  The micropump M is composed of the housing 100 and the lid member 200 as follows. 8 and 9, the block-shaped housing 100 has an annular groove 1b having a semicircular cross section formed on a flat upper surface 1a. As a material of the housing 100, a glass substrate, a silicon substrate, PDMS (poly-dimethylsiloxane), a ceramic substrate, or the like can be used. When the groove 1b is formed on a glass substrate, a film is formed on the glass substrate by spin coating a photo-curing resin or thermosetting resin, resists, polyimide, etc., and then subjected to exposure, development, etching, and the like. A minute groove is produced. In addition, the substrate body may be manufactured using various processing techniques for etching the flow path using wet etching of the substrate body, dry etching by RIE, or the like.

  On the other hand, the plate-like lid member 200 that can be formed of the same material as that of the housing 100 is formed with an annular groove 2b having a semicircular cross section corresponding to the groove 1b on the flat lower surface 2a. When the lid member 200 is overlaid on the housing 100, the annular circulation path R is formed by the grooves 1b and 2b facing each other.

  The lid member 200 has a function of bonding the housing 100 to the housing 100 using an adhesive, hydrofluoric acid, anodic bonding, mechanical fixation, etc., and isolating the circulation path R from the outside air. If it is not necessary to flow a high-pressure fluid, there is no need to fix the machine. In addition, the linear supply path I and the discharge path O are formed in a similar manner so as to be connected in a tangential direction with respect to the circulation path R, and open to the outside.

  In this circulation path R, for example, 11 non-magnetic microspheres 300 and one magnetic microsphere 400 having diameters larger than the supply path I and the discharge path O, respectively, roll. Arranged freely. As the microsphere 400, an iron ball containing a ferromagnetic material or the like may be used, and various plating, vapor deposition, surface treatment processing, or the like may be performed in order to protect the surface. As the microsphere 300, a metal other than a magnetic material, resin, ceramic, or the like can be used. The diameter of the microspheres 300 and 400 can be reduced to the order of several μm.

  On the upper surface of the lid member 200, for example, six electromagnets 5 </ b> A to 5 </ b> F are arranged along the circulation path R at equal intervals in the circumferential direction. The electromagnets 5A to 5F are selectively driven and excited by the drive circuit DR.

  The operation of this embodiment will be described. FIGS. 10A to 10C are views similar to FIG. 9 showing the operation of the micropump M. FIG. The supply path I is connected to a fluid supply source, and the discharge path O is connected to a fluid supply section. First, in FIG. 10A, the drive circuit DR (FIG. 8) detects the position of the magnetic microsphere 400 (shown by hatching) by a sensor (not shown), and the electromagnet 5A separated in the clockwise direction therefrom. Select to excite. Then, the microsphere 400, which is a magnetic body, is biased by the magnetic force generated from the electromagnet 5A and tries to move clockwise. Since the remaining microspheres 300 are not affected by the magnetic force, they are pushed by the microspheres 400 and move in the same direction. When all the microspheres 300 and 400 in the circulation path R rotate and move in the same direction, the fluid inside the microspheres 300 and 400 moves in the same direction. Thereby, the fluid taken in from the supply path I can be discharged from the discharge path O.

  Subsequently, in FIG. 10B, when the drive circuit DR (FIG. 8) detects that the magnetic microsphere 400 has approached the electromagnet 5A, it stops exciting the electromagnet 5A and rotates it clockwise. The electromagnet 5B adjacent in the direction is selected and excited. Then, by the magnetic force generated from the electromagnet 5B, the microsphere 400, which is a magnetic body, is urged in the same direction, and tries to move further clockwise with the other microsphere 300 after passing through the electromagnet 5A.

  Further, in FIG. 10C, when the drive circuit DR (FIG. 8) detects that the magnetic microsphere 400 has approached the electromagnet 5B, the drive circuit DR stops the excitation of the electromagnet 5B and then rotates clockwise. The adjacent electromagnet 5C is selected and excited. Then, the magnetic sphere 400 is biased in the same direction by the magnetic force generated from the electromagnet 5 </ b> C, and passes through the electromagnet 5 </ b> B to move further clockwise with the other microspheres 300. By repeating the above control, the microspheres 300 and 400 can be continuously rotated and fluid can be continuously pumped.

  FIGS. 11A to 11C are views similar to FIG. 9 showing the operation of the micropump according to another embodiment. In the present embodiment, for example, ten non-magnetic microspheres 300 and two magnetic microspheres 400 in a 180 degree phase are arranged in the circulation path R so as to roll freely. Since other configurations are the same as those in the above-described embodiment, the description thereof is omitted.

  The operation of the present embodiment will be described. In FIG. 11A, the drive circuit DR (FIG. 8) detects the positions of the two microspheres 400, 400 of the magnetic material by a sensor (not shown), The electromagnets 5A and 5D separated in the direction are selected and excited. Then, the microspheres 400 and 400, which are magnetic bodies, are urged by the magnetic force generated from the electromagnets 5A and 5D, and try to move clockwise. Since the remaining microspheres 300 are not affected by the magnetic force, they are pushed by the microspheres 400 and move in the same direction. When all the microspheres 300 and 400 in the circulation path R move in the same direction, the fluid inside of the microspheres 300 and 400 moves in the same direction. Thereby, the fluid taken in from the supply path I can be discharged from the discharge path O.

  Subsequently, in FIG. 11B, when the drive circuit DR (FIG. 8) detects that the magnetic microspheres 400 and 400 approach the electromagnets 5A and 5D, the drive circuit DR (FIG. 8) stops the excitation of the electromagnets 5A and 5D. Then, the electromagnets 5B and 5E adjacent in the clockwise direction are selected and excited. Then, the magnetic spheres 400 and 400, which are magnetic bodies, are urged in the same direction by the magnetic force generated from the electromagnets 5B and 5E, and pass through the electromagnets 5A and 5D to further move clockwise with the other microspheres 300. .

  Further, in FIG. 11C, when the drive circuit DR (FIG. 8) detects that the magnetic microspheres 400 and 400 approach the electromagnets 5B and 5E, the drive circuit DR (FIG. 8) stops the excitation of the electromagnets 5B and 5E. Then, the electromagnets 5C and 5F adjacent in the clockwise direction are selected and excited. Then, the magnetic spheres 400 and 400, which are magnetic bodies, are urged in the same direction by the magnetic force generated from the electromagnets 5C and 5F, and pass through the electromagnets 5B and 5E to move further clockwise with the other microspheres 300. . By repeating the above control, the microspheres 300 and 400 can be continuously rotated and fluid can be continuously pumped. According to the present embodiment, since the two microspheres 400 are simultaneously energized, the rotational speed is approximately doubled, and high-speed pumping of fluid is possible.

  FIG. 12 is a view similar to FIG. 8 showing a modification of the present embodiment. In the modification shown in FIG. 12A, the annular groove 1 b is formed only on the upper surface 1 a of the housing 100, and the annular groove is not formed on the lower surface of the lid member 200. On the other hand, in the modification shown in FIG. 12B, the annular groove 2 b is formed only on the lower surface 2 a of the lid member 200, and the annular groove is not formed on the upper surface of the housing 100.

  Further, in the modification shown in FIG. 12C, the electromagnets 5 </ b> A to 5 </ b> F are arranged on the lower surface of the thin housing 100 instead of the electromagnets 5 </ b> A to 5 </ b> F on the upper surface of the lid member 200. Moreover, in the modification shown in FIG.12 (d), instead of arrange | positioning the electromagnets 5A-5F on the upper surface of the cover member 200, the recessed parts 1c and 2c are formed in the side surface of the housing 100 and the cover member 200 over the perimeter, Electromagnets 5A to 5F are embedded and arranged in an annular space formed by the recesses 1c and 2c at the time of assembly. Further, in the modification shown in FIG. 12 (e), instead of arranging the electromagnets 5A to 5F on the upper surface of the lid member 200, the electromagnets 5A to 5F are opposed to the side surfaces of the housing 100 and the lid member 200, and external electromagnets are disposed outwardly. 5A to 5F are arranged.

  FIG. 13 is a plan view of a flow path substrate according to the third embodiment. A flow path substrate 1 ′ in FIG. 13 is provided with a micropump M similar to that in FIGS. 8 and 9 described above to flow a solution in the flow path 23. That is, as shown in FIG. 13, a recess 2 similar to that shown in FIGS. 1A and 1B is formed on the support 11, and a groove-shaped delivery path 28 and discharge path 29 are formed in the same manner as the recess 2. The discharge path 28 is connected to one solution tank 21a, the discharge path 29 is connected to the other solution tank 21b, and when the micropump M is formed, the discharge path O of the micropump M is connected to the delivery path 28. The supply path I is connected to the discharge path 29. In addition, the circulation path R of FIG. 8, FIG. 9 comprises a pump flow path.

  A groove 1b is formed in the same manner as the recess 2 on the support 11 in FIG. 1B instead of the housing 100 in FIGS. 8 and 9, and a lid member 200 is formed on the support 11 as shown in FIG. 12A. By covering and covering the lid member 200 with the electromagnets 5A to 5F, the flow path substrate 1 ′ having the micropump M can be obtained. By operating the micropump M as shown in FIGS. 10A to 10C, the solution can flow in the flow path 23 from one solution tank 21a toward the other solution tank 21b. The flow path substrate 1 'can be used in the reaction apparatus of FIG.

  In FIG. 13, connection means for connecting the electromagnets 5A to 5F (FIG. 8) of the micropump M and the drive circuit DR (FIG. 8) can be separately provided. In addition, the flow path substrate 1 ′ includes a single recess 2, but may include a plurality of recesses 2. Further, the flow path substrate 1 ′ may be provided with a cooling unit such as a Peltier element as in the second embodiment.

  Further, as a processing method for forming the above-described micropump on a support or the like, one of the inventors of the present invention previously proposed an etching process proposed in Japanese Patent Application No. 2006-195501, a process using a film material, and a mold. A molding process using can be used.

  <Fourth embodiment>

  In the fourth embodiment, when a sample solution is caused to flow in a flow path of a flow path substrate using centrifugal force, and the separation or synthesis is performed with the sample solution, the reaction process can be observed on a monitor.

  FIG. 14 is a perspective view showing an example of the overall configuration of a visible centrifuge according to the fourth embodiment. FIG. 15 is a partial configuration diagram for explaining the internal configuration of the fixed component of FIG. 16 is a top view of an example of the overall configuration of the visible centrifuge shown in FIG. 14 as viewed from above. FIG. 17 is a longitudinal sectional view of the observable centrifugal apparatus in a state where the spindle unit is mounted on the rotating disk of FIG. FIG. 18 is an overall perspective view of the observable centrifugal apparatus with the air spindle unit 628 mounted as the spindle unit.

  Hereinafter, a visible centrifugal device according to a fourth embodiment will be described with reference to the accompanying drawings. 14 to 18 show a visible centrifuge (hereinafter also simply referred to as “apparatus”) AP according to the present embodiment, and the apparatus AP rotates around a predetermined rotation axis 602. The turntable 604, the reaction flow path substrate 1 that is disposed on the turntable 604, accommodates a sample (sample solution) and rotates together with the turntable 604, and the state of the sample in the flow path substrate 1 are visually observed. A microscope 608 is provided. Then, the device AP applies a centrifugal force to the sample in the flow path substrate 1 to separate or synthesize a predetermined substance (liquid, solid and gas, or a mixture thereof) from the sample. Yes.

  Note that the size and shape of the apparatus AP, specifically, the size and shape of the turntable 604 may be arbitrarily set according to the nature and number of samples to be centrifuged or centrifugally synthesized. In FIG. 2, a case where the rotating disk 604 is configured as a disk having a diameter of 220 mm is assumed as an example.

  Further, the flow path substrate 1 can accommodate a predetermined sample therein, and can cause the sample to undergo a centrifugal separation reaction or a centrifugal synthesis reaction, and has the same configuration as in FIGS. 1 (a), (b), and FIG. The flow path 23 is formed along the plane. The flow path substrate 1 is fixed with respect to the turntable 604 in a state in which the sample is accommodated therein, and rotates with the turntable 604 so that the sample (sample solution) can flow in the flow path 23 by centrifugal force. . The flow path substrate 1 may be configured as shown in FIGS. 3 and 4, and may be used in combination with an electrode and centrifugal force for sample solution flow. Either the electrode or the centrifugal force may be selected according to the characteristics. Furthermore, an electrode for flowing the sample solution may be omitted. Moreover, although the flow path 23 is single, multiple may be sufficient.

  In the apparatus AP, the microscope 608 is fixed to a predetermined position of the rotating plate 604 so that the state of the sample in the channel substrate 1 can be observed, and the rotating plate 604 includes a channel substrate captured by the microscope 608. An imaging device 610 for capturing a microscopic image of a sample state in 1 and a video wireless transmission device 612 for wirelessly transmitting a captured image of the microscope image captured by the imaging device 610 as a moving image in real time are attached. ing. In this embodiment, as an example, the microscope 608 has an objective lens 608a that captures the state of the sample in the flow path substrate 1, and an optical path for transmitting the microscope image captured by the objective lens 608a to the imaging device 610. And a lens barrel 608b formed inside. In addition, in the present embodiment, various imaging devices capable of capturing a microscopic image of the sample state in the flow path substrate 1 captured by the microscope 608 can be applied to the imaging device 610. A case where a CCD camera is applied as the imaging device 610 is assumed as an example.

  In this case, in the microscope 608, the optical path of the microscope 608 is partially refracted at a predetermined angle with respect to the surface of the rotating plate 604 (the upper surface in FIG. 17) 604a in the lens barrel 608b, and the imaging device ( The CCD camera (hereinafter referred to as a CCD camera) 10 is positioned in the vicinity of the rotation center of the turntable 604 so that the above-described microscope image can be taken on the optical path of the microscope 608 refracted at the predetermined angle. In the following description, the above-described optical path of the microscope 608 is referred to as an observation optical path, and light traveling along the observation optical path is referred to as observation light.

  In the present embodiment, as an example, as shown in FIG. 17, the mirror 614 is tilted with respect to the observation optical path by a predetermined angle arbitrarily set according to the refraction angle of the observation optical path into the lens barrel 608b of the microscope 608. Are arranged. In the configuration shown in FIG. 17, the apparatus AP is configured such that the objective lens 608a of the microscope 608 captures the sample state in the flow path substrate 1 from above in the vertical direction (the vertical direction in the figure), and the rotation center of the turntable 604. The microscope camera 610 captures a microscope image captured by the objective lens 608a from a direction (horizontal direction) parallel to the surface 604a of the turntable 604 in the vicinity. For this reason, the mirror 614 is tilted backward at an angle of about 135 ° with respect to the entrance direction of the observation light and disposed in the lens barrel 608b of the microscope 608 so that the entered observation light is refracted by about 90 °. It is further advanced so as to be parallel to the disk surface 604a of the rotating disk 604. Note that the microscope 608 may have a configuration in which an observation optical path refracted at a substantially right angle is formed in the lens barrel 608b by refracting the lens barrel 608b at a substantially right angle.

  In this case, by positioning the microscope 608 at the peripheral edge of the rotating disk 604, the observation light entering from the vertical direction is parallel to the surface 604a of the rotating disk 604 from the circumferential direction of the rotating disk 604 toward the center by the mirror 614. The traveling direction can be changed so as to be refracted. As a result, the microscope 608 has a structure in which the observation light (that is, the microscope image) reaches (converges) toward the center of the rotating disk 604, that is, the direction of the rotation center of the rotating disk 604, and observes the CCD camera 610. By positioning at the destination (convergence) of the optical path, the CCD camera 610 can capture the observation light of the microscope in the vicinity of the rotation center of the turntable 604. Specifically, the microscope image can be taken. be able to.

  For this reason, the CCD camera 610 can be positioned in the vicinity of the rotation center of the turntable 604, and even if a centrifugal force is generated by the rotation of the turntable 604, the centrifugal force acting on the CCD camera 610 is achieved. The CCD camera 610 is not hindered by the centrifugal force and the microscope image at the time of shooting is not blurred, and the CCD camera 610 can always take a stable microscope image. Become.

  Further, as described above, the microscope 608 has a structure in which the observation optical path is refracted by the mirror 614, whereby the height of the lens barrel 608b of the microscope 608 (the vertical distance in FIG. 17) can be suppressed. Thereby, even if rotational vibration is generated by rotating the turntable 604, the rigidity of the microscope 608 with respect to the rotational vibration can be increased, and the sample state in the flow path substrate 1 that is always stable in the microscope 608 can be improved. Observation can be performed. However, in order to suppress the height of the lens barrel 608b, it is preferable that the microscope 608 has a structure in which the refraction angle of the observation optical path is greater than 0 ° and 90 ° or less.

  The microscope 608 has a structure in which the lens barrel 608b can move up and down in the vertical direction (vertical direction (up and down direction in FIG. 17)) with respect to the flow path substrate 1. Thus, the distance (focal length) between the flow path substrate 1 (specifically, the sample) and the objective lens 608a can be adjusted. In this case, the rotating disk 604 is provided with a guide (hereinafter referred to as a Z-axis guide) 620 extending in a vertical direction with respect to the disk surface 604a, and the lens barrel 608b is attached to the Z-axis guide 620. The microscope 608 has a structure for adjusting the focal length between the sample and the objective lens 608a.

  In the present embodiment, the objective lens 608a of the microscope 608 and the flow path substrate 1 are accommodated in the same component (hereinafter referred to as a fixed component) 616, and the accommodated objective lens 608a and the flow path substrate 1 are accommodated. Is fixed to the rotating disk 604 integrally with the fixed component 616, whereby relative displacement due to external vibration (specifically, rotational vibration generated by rotation of the rotating disk 604) between the objective lens 608a and the flow path substrate 1 is achieved. Is minimized. Thereby, the microscope 608 can always stably capture the sample state in the flow path substrate 1 as a clear image without blurring, and can accurately and reliably observe the state of the sample in the separation process or the synthesis process. It becomes possible.

  In this case, as shown in FIG. 15, a predetermined illumination device (for example, an edge-type LED (Light Emitting Diode) backlight) 622 is provided inside the fixed component 616, and the sample is taken as an object of the microscope 608. Observation is possible with the illumination device 622 illuminating and transmitting from the side opposite to the lens 608a. Thereby, the state of the sample in the flow path substrate 1 can be observed more clearly, and the sample state can be captured as a clearer microscope image by the objective lens 608a. Note that the LED 22a, which is the light source of the illumination device (edge-type LED backlight) 22, rotates the rotating disk 604 in order to reduce the effect of centrifugal force generated by the rotation of the rotating disk 604, as with the CCD camera 610 described above. It is positioned near the center.

  Here, the specific configuration of the lighting device 622 is not particularly limited as long as the lighting device 622 can observe the sample while illuminating and transmitting the sample. For example, an arbitrary illumination device may be selected according to the nature or type of the sample. As an example, in this embodiment, LED illumination (edge type backlight) MEBL-CW25 manufactured by Moritex Corporation is used. ing. However, for example, a lighting device having performance equivalent to or higher than that of the lighting device 622 may be used.

  The fixed component 616 adjusts the focal distance between the objective lens 608a of the microscope 608 and the sample, and is set to an appropriate distance, specifically, the relative position between the objective lens 608a and the sample, specifically, the objective lens 608a. The height of the sample is fixed. Accordingly, the height of the objective lens 608a of the microscope 608 relative to the sample can be kept constant during the separation reaction or the synthesis reaction, and the state of the sample can be observed stably.

  In the present embodiment, a camera image (captured image) of the microscope image captured by the CCD camera 610 is converted into a moving image in real time together with the CCD camera 610 that captures the above-described microscope image as a moving image with respect to the turntable 604. A video wireless transmission device 612 for wireless transmission is attached. As described above, by adopting a wireless system instead of a wired system as a system for transmitting an image captured by the CCD camera 610 to an external receiver (not shown), the CCD camera 610 and the video wireless system together with the turntable 604 are used. Even when the transmission device 612 is rotated, there is no need to take out signal lines directly from them, and there is no need to consider handling of signal lines. As a result, the peripheral structure of the CCD camera 610 and the wireless video transmission device 612 can be easily simplified.

  In addition, since it is not necessary to consider the handling of the signal line, the CCD camera 610 and the video wireless transmission device 612 are rotated together with the rotating plate 604 (specifically, the microscope 608 and the sample in the flow path substrate 1). The microscope image can be taken by the CCD camera 610 at an arbitrary high frame rate (frame number) without being restricted by the number of rotations of the turntable 604, and the camera image of the taken microscope image can be externally transmitted. It can be transmitted to a receiving device (not shown). Thereby, by providing a display such as a liquid crystal panel or a CRT (Cathode Ray Tube) display as such an external receiving device, the above-described camera image (that is, the state of the sample inside the flow path substrate 1) can be obtained. A sample separation reaction or synthesis reaction can be advanced while confirming in real time on such a display. In addition, by analyzing the camera video recorded via a personal computer, the behavior of the sample (specifically, its internal substances, separated substances, synthetic substances, etc.) is monitored, and the optimum rotation conditions of the turntable 604 are monitored. (Another way of understanding is to estimate the optimum magnitude of the centrifugal force acting on the sample) and to control the rotation of the turntable 604 with the estimated optimum conditions (for example, rotation speed and rotation time). Is also possible.

  In this case, similarly to the CCD camera 610 described above, the video wireless transmission device 612 is positioned in the vicinity of the rotation center of the turntable 604 in order to reduce the action of centrifugal force generated by the rotation of the turntable 604. Video data of a camera image taken by the camera 610 is wirelessly transmitted from a predetermined antenna 618 to an external receiver (a display device such as a liquid crystal panel or a CRT display connected to the receiver). Similarly, the antenna 618 is also configured to rise in the vicinity of the rotation center of the turntable 604, specifically, on an extension line of the rotation center of the turntable 604, in order to reduce the action of centrifugal force generated by the rotation of the turntable 604. Yes.

  When the video wireless transmission device 612 wirelessly transmits the video data (video signal) of the camera video to an external receiver (not shown), the video data transmission speed (bit rate) and data format (frequency, compression / non-compression) The presence or absence, etc.) may be arbitrarily set according to the usage mode and usage conditions of the device AP. For example, in the present embodiment, as an example, a camera image of a microscope image taken by the CCD camera 610 is converted into video data of an uncompressed digital signal having a frequency of 2.4 GHz by the video wireless transmission device 612, and the video data Is transmitted wirelessly to an external receiver. As a result, the video signal transmitted from the video wireless transmission device can be transmitted to the external receiving apparatus without being lost, and the separation reaction can be performed while confirming the sample state with a clear and stable video in the external receiving apparatus. Alternatively, the synthesis reaction can proceed. However, the video data transmitted from the video wireless transmission device 612 to the external reception device may be a compressed signal or an analog signal instead of the above-described uncompressed digital signal.

  Here, as long as the CCD camera 610 can take a microscopic image of the sample state in the flow path substrate 1 captured by the microscope 608, the specific configuration thereof is not particularly limited. For example, an arbitrary CCD camera may be selected according to the usage mode and usage conditions of the apparatus AP. As an example, in this embodiment, a color board camera MSC-90 manufactured by MOSWELL Co., Ltd. is used. . However, for example, a CCD camera having performance equivalent to or higher than that of the CCD camera 610 may be used.

  The specific configuration of the video wireless transmission device 612 is not particularly limited as long as it can wirelessly transmit a camera image of a microscope image taken by the CCD camera 610 to an external receiving device (not shown). . For example, an arbitrary video wireless transmission device may be selected according to the usage mode or usage conditions of the apparatus AP. As an example, TRX24mini manufactured by Aiden Videotronics Co., Ltd. is used in the present embodiment. However, for example, a video wireless transmission device having performance equivalent to or higher than that of the video wireless transmission device 612 may be used.

  Note that various electrical components provided in the apparatus AP, such as the above-described CCD camera 610, video wireless transmission device 612, and illumination apparatus 622, may be a predetermined power supply device (for example, a battery), as shown in FIGS. 624). In this case, the power supply device 624 supplies power capable of normally operating such various electrical components (such as the CCD camera 610, the video wireless transmission device 612, and the illumination device 622) during the separation reaction or the synthesis reaction for the sample. As long as it can be supplied stably, its specific configuration is not particularly limited. For example, an arbitrary power supply device may be selected according to the magnitude of power required for the various electrical components described above. As an example, in the present embodiment, UBBP01 (a battery made by ULTRAP LIFE Co., Ltd.) A voltage of 3.7 V and a battery capacity of 1.8 APh) are used. However, for example, a power supply device having performance equivalent to or higher than that of the power supply device 624 may be used.

  In the present embodiment, four such power supply devices (batteries) 624 are used in series, and these four batteries 624 are moved to positions symmetrical with respect to the rotation center of the turntable 604 (with a phase difference of 180 °). 2) Evenly arranged two by two, and arranged with a phase difference of 90 ° with respect to the microscope 608, the flow path substrate 1 and the fixed component 616 (see FIG. 16). In this case, as an example, the battery 624 is embedded in a mounting portion formed by recessing the disk surface 604 a of the rotating disk 604, fixed by a plate-like member 626, and attached to the rotating disk 604.

  Here, in the apparatus AP, the rotating shaft 602 of the turntable 604 is rotated by a predetermined driving device (for example, a spindle motor) (not shown) and is rotatably supported by various bearings 627. FIG. 17 shows a configuration in which the rotating shaft 602 is supported by a rolling bearing 627 using balls as rolling elements, as an example. In this case, the rolling bearing 627 may be a roller bearing in which various rollers (cylindrical rollers, tapered rollers, spherical rollers, etc.) are applied as rolling elements, in addition to various ball bearings in which balls are applied as rolling elements. In the configuration shown in FIG. 17, the rotation shaft 602 is supported by two bearings 627. However, the rotation shaft 602 may be supported by one bearing 627 or by three or more bearings 627. You may support.

  Note that an air bearing is applied as the bearing 627 in place of the above-described various rolling bearings, and the rotary shaft 602 is rotatably supported by the air bearing, so that the rotational vibration generated when the rotating disk 604 rotates is remarkably increased. More preferably, the rotational vibration of the microscope 608 and the CCD camera 610 can be suppressed, and stable observation and photographing of the sample state during the separation reaction or the synthesis reaction can be performed. Here, as an example, the air bearing has a structure in which the rotary shaft 602 is rotatably supported by a cylindrical housing positioned so as to cover the outer peripheral surface of the rotary shaft 602 over the entire circumference. Air is blown toward the outer peripheral surface of the rotating shaft 602 from a plurality of outlets (spout holes) provided on the peripheral surface (the surface facing the outer peripheral surface of the rotating shaft 602), and the inner peripheral surface of the housing and the outer peripheral surface of the rotating shaft 602 Is kept in a non-contact state, the rotating shaft 602 can be rotated very smoothly.

  Further, in the present embodiment, the rotary shaft 602 and the bearing 627 that rotatably supports the rotary shaft 602 are configured as a spindle unit 628 in which these are integrated with the housing, and the spindle unit 628 is attached to the rotary disc 604. As a result, the turntable 604 is configured to rotate about the rotation shaft 602. In this case, the central portion of the turntable 604 is convex with a predetermined size toward the upper side (the side on which the microscope 608, the flow path substrate 1, the CCD camera 610, the video wireless transmission device 612, etc. are disposed). And a spindle unit mounting portion 604b formed by projecting the lower side of the rotating plate 604 (the side opposite to the side on which the above-described components are disposed) into a convex shape. 628 is inserted into the spindle unit attachment portion 604b from the lower side of the turntable 604 and attached to the turntable 604.

  As described above, the microscope AP 608, the flow path substrate 1, the CCD camera 610, the video wireless transmission device 612, and the like are provided by configuring the apparatus AP so that the rotating disk 604 covers the spindle unit 628. The distance between the rotational center of gravity during rotation of the turntable 604 and the shaft support portion in which the rotation shaft 602 is rotatably supported by the bearing 627 can be reduced, and the rotational moment generated in the shaft support portion can be effectively reduced. it can.

  In addition, in this Embodiment mentioned above, although it did not mention in particular about the material of the structural member of apparatus AP, if various materials are arbitrarily selected and used according to the usage condition, purpose, etc. of apparatus AP, it uses. Good. As an example, in the present embodiment, the material of the turntable 604 and various attachment members for attaching the microscope 608, the channel substrate 1, the CCD camera 610, the video wireless transmission device 612, and the like to the turntable 604. Is made of high-strength APl alloy (A2017), so that the weight of these members is reduced while sufficiently securing rigidity during rotation.

  Further, in the present embodiment, in order to equalize the weight balance of the apparatus AP during rotation and reduce the run-out stress on the spindle unit 628 that occurs during rotation, the microscope 608 and the flow path substrate 1 are in contact with the rotating plate 604. A predetermined balance weight 630 is provided at a position that is substantially symmetrical with respect to the arrangement position and the rotation center (on the opposite side to the rotation center). The weight of the balance weight 630 and the position of the balance weight 630 are the weights and balances (center of gravity) of various members such as the microscope 608, the flow path substrate 1, the CCD camera 610, and the video wireless transmission device 612 disposed on the turntable 604. In accordance with the above, adjustment may be made so that the above-described swinging stress on the spindle unit 628 is reduced.

  In the apparatus AP, when the separation reaction or synthesis reaction of the sample is observed with higher accuracy, an air spindle unit in which the rotary shaft 602 is rotatably supported by the above-described air bearing is used as the rotary disk 604 as the spindle unit 628. It is good also as a structure with which it mounts | wears. As a result, the rotational vibration generated when the turntable 604 rotates can be remarkably reduced, and the sample state during the separation reaction or the synthesis reaction can be observed with a more stable high-quality camera image. . In this case, as the air spindle unit, for example, GBS100H manufactured by NSK Ltd. can be used.

  Next, the operation and effect of driving and flowing fluid by applying centrifugal force generated by mounting and rotating the flow path substrate 1 to the visible centrifuge AP as described above to the sample (sample solution) will be described. .

  Fluid connection with an external device to the flow path substrate 1 (when using a pump such as that shown in FIGS. 8 to 13 for fluid drive) and electrical connection (such as electroosmotic flow as shown in FIG. 1 for fluid drive) Is unnecessary, and the structure of the flow path substrate 1 can be simplified. As an effect, handling of the flow path substrate 1 is facilitated, automation is facilitated, and analysis speed is improved. Further, the flow path substrate 1 can be further reduced in size, and analysis with a smaller sample becomes possible. In this case, since the cells cannot be crushed electrically, they are crushed by mechanical collision.

  In addition, since the peripheral device can be reduced in size, the entire measurement system can be reduced in size.

  Further, the fluid can be driven without being affected by the chemical characteristics of the sample. In particular, it is possible to drive (analyze) even a sample mainly composed of a solution that is easily electrolyzed by applying an electric field. Moreover, it can be used without worrying about these effects even for samples that may change due to electrical stimulation, and the application range is widened.

  In addition, the centrifugal effect of the sample can be generated at the same time, and separation by the specific gravity of the sample is possible.

  Furthermore, by using the visible centrifuge of this embodiment, the detection status can be ascertained even if the reaction is in the region of low rotational speed (low centrifugal force) without missing information (dropping frames). can do.

  Next, a configuration in which an epi-illumination device that illuminates the sample from above is added to the visible centrifuge AP of FIGS. 14 to 18 will be described with reference to FIG. FIG. 19 is a longitudinal sectional view similar to FIG. 17 showing a configuration in which an epi-illumination device is added to the visible centrifuge AP.

  When observing a substance with the same optical transmittance as the background (transparent), such as cells and glass beads in solution, it is difficult to observe the contrast due to the shape with the backlight illumination. is there. As a countermeasure against this, an epi-illumination device is provided in the visible centrifuge AP as shown in FIG.

  That is, the mirror (614) on the upper side of the objective lens (608a) is a half mirror (614a), and the LED illumination unit (640) is provided above the half mirror (614a) to constitute a coaxial (epi-illumination) illumination device. Illumination light m from the LED illumination unit 640 passes through the half mirror 614a and irradiates the sample in the flow path substrate 1 through the objective lens 608a. The reflected light n from the sample is reflected by the half mirror 614a through the objective lens 608a and reaches the CCD camera 610.

  For example, a commercially available high-brightness green LED (100047 series manufactured by OPTSOURCE, φ3 mm, luminous intensity 6800 mcd) can be used for the LED illumination unit 640, but the color tone and luminance can be selected depending on the target sample. In addition, when there is a concern about the breakage of the LED due to the centrifugal strength (number of rotations, time), a light source (LED) can be arranged near the rotation center and guided to the microscope with an optical fiber.

  According to the configuration in which the epi-illumination device of FIG. 19 is added, by observing the reflected light from the sample in the flow path substrate 1, even if the optical transmittance is about the background and the circumference, the shadow (contrast) due to the reflection of the sample surface. Therefore, the behavior of the sample can be clearly observed.

  Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the examples.

  In this embodiment, the visible centrifuge AP is used for selecting the size of polystyrene beads. That is, a channel substrate having a dumbbell-shaped channel (a channel having a shape in which circular solution tanks are provided at both ends and the solution tanks are connected by a straight channel) as shown in FIG. A polystyrene bead solution having a different size from the anti-centrifugation side was introduced in a state where the channel substrate was filled with the solution, and the speed at which the beads passed through the channel by centrifugal force was measured.

  The flow path substrate has a structure in which a PDMS resin having a fine flow path pattern as shown in FIG. 20 is attached to a glass substrate. The flow path pattern is a shape in which circular solution layers having a diameter of 3 mm are connected by a straight flow path having a width of 700 μm and has a depth of about 120 μm. A solution introduction hole is formed on one side of the solution layer, from which a bead solution can be introduced.

  The solution introduction hole is set on the anti-centrifugation side and attached to the visible centrifuge AP so that the linear flow path portion can be observed with a CCD camera. At this time, the flow path is filled with a test solution (0.1 M mannitol aqueous solution). When the polystyrene solution is introduced from the solution introduction hole and the centrifuge under visible light AP is driven, the polystyrene beads are moved to the centrifugal solution tank through the linear flow path by the centrifugal force. The visible centrifuge AP can measure the moving speed v of the polystyrene beads accurately because the moving speed of the polystyrene beads can be measured at an arbitrary number of rotations (centrifugal force).

  Using polystyrene beads having a diameter of 10 μm and a diameter of 40 μm (Mortex Corp. 4000 series) as samples, the difference in moving (sedimentation) speed v under each centrifugal force was measured. The measurement results are shown in FIG. As can be understood from FIG. 21, it was demonstrated that beads having a larger diameter have a higher moving speed and that size selection can be performed while observing the behavior of each bead.

  As described above, the best modes and examples for carrying out the present invention have been described, but the present invention is not limited to these, and various modifications are possible within the scope of the technical idea of the present invention. Such modifications are also within the scope of the present invention.

FIG. 2A is a plan view of a flow path substrate according to the first embodiment, and FIG. 2B is a diagram illustrating a stacked configuration of the flow path substrate of FIG. It is the II sectional view taken on the line of Fig.1 (a). It is a figure for demonstrating the effect in 1st Embodiment ((a) is the state which crushes a cell, (b) shows the state which isolate | separates each molecule | numerator after crushing.) It is a figure which shows the other structural example of the structure in 1st Embodiment. It is a functional block diagram of the reaction apparatus of 2nd Embodiment. FIG. 6 is a diagram (a) is a front view, (b) is a plan view, and (c) is a side view) showing the appearance of the reaction apparatus of FIG. It is a figure which shows schematic structure of the Peltier device used by 2nd Embodiment. It is side surface sectional drawing of the micropump concerning 3rd Embodiment. It is the figure which cut | disconnected the structure of FIG. 8 by the II-II line | wire, and looked at the arrow direction. It is a figure similar to FIG. 9 which shows operation | movement (a)-(c) of a micropump. It is a figure similar to FIG. 9 which shows operation | movement of the micro pumps (a)-(c) concerning another embodiment. It is a figure similar to FIG. 8 which shows the modification (a)-(e) of the micropump of FIG. It is a top view of a channel substrate by a 3rd embodiment. It is a perspective view which shows the example of whole structure of the visible centrifuge which concerns on 4th Embodiment. It is a partial block diagram for demonstrating the structure inside the fixed component of FIG. It is the top view which looked at the example of whole composition of the centrifuge under visible condition of Drawing 14 from the top. FIG. 15 is a longitudinal sectional view of the observable centrifugal apparatus in a state where a spindle unit is mounted on the rotating disk of FIG. 14. FIG. 2 is an overall perspective view of the observable centrifugal apparatus with an air spindle unit 628 mounted as a spindle unit. It is the same longitudinal cross-sectional view as FIG. 17 which shows the structure which added the epi-illumination apparatus to visible centrifuge AP. It is a top view which shows roughly the flow-path pattern of the flow-path board | substrate used in the present Example. It is a graph which shows the relationship between the centrifugal acceleration when a polystyrene bead from which a diameter differs in a present Example moves in a linear flow path, and a moving speed.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Flow path board | substrate 2 Recessed part 4 Filter 45 Structure 6 Cell crushing part 11 Support body 12 Photosensitive resin layer 21 Solution tank 23 Flow path 31 Peltier element 33 Water cooler 45 Structure 51 Driving electrode 61 Cell trapping part 62 Crushing electrode 1 'Channel substrate M Micropump C Cell C1 Inclusion C2 Cell membrane AP Visible centrifuge

Claims (20)

In the flow path substrate that is formed so that the flow path extends along the surface and moves the cells in the solution introduced into the flow path in the flow path,
A flow path substrate comprising a filter in the middle of the flow path for separating inclusions and dust of cells moving in the flow path according to their molecular size. A cell crushing unit for crushing cells contained in the solution on the upstream side of the filter,
The flow path substrate according to claim 1, wherein the filter separates inclusions in a solution that moves in the flow path after the cells are crushed by the cell crushing unit according to the size.   The flow path substrate according to claim 1 or 2, wherein the filter prevents passage of substances other than inclusions to be separated and extracted from inclusions having a predetermined size or more.   4. The flow path substrate according to claim 1, wherein the filter includes at least one structure that divides a flow path section into a plurality of sections. 5.   The flow path substrate according to claim 4, wherein the structure is a columnar member extending in an out-of-plane direction from a bottom surface of the flow path.   The flow path substrate according to claim 5, wherein the filter includes a plurality of structures arranged side by side in the flow path width direction.   The channel substrate according to claim 5 or 6, wherein the structure has a shape in which a portion facing the upstream side of the channel projects from the downstream side toward the upstream side.   The pair of drive electrodes for moving the sample molecules in the flow path are integrally formed on the upstream side and the downstream side of the flow path, respectively. The flow path substrate according to Item.   9. The flow path substrate according to claim 1, wherein the flow path is formed by partially laminating a photosensitive resin layer on a plate-like support.   The flow path substrate according to any one of claims 1 to 9, further comprising a cooling unit that cools the flow path substrate.   The flow path substrate according to claim 10, wherein the cooling unit is a Peltier element.   The flow path substrate according to claim 11, wherein the Peltier element is disposed so that a heat absorption surface thereof is in contact with a bottom surface of the flow path substrate.   The flow path substrate according to claim 11 or 12, further comprising a second cooling unit that cools a heat dissipation surface of the Peltier element.   The flow path substrate according to claim 1, further comprising a micropump for moving the cells in the flow path.   The micropump includes a magnetic object and a non-magnetic object movably disposed in a pump flow path communicating with the flow path, and a plurality of electromagnets disposed along the pump flow path. By supplying electric power to an electromagnet close to the magnetic object, the magnetic object is moved together with the non-magnetic object in a predetermined direction in the pump flow path by the generated magnetic force. The channel substrate according to claim 14, wherein cells in the solution are moved in the channel. A reaction apparatus comprising the flow path substrate according to any one of claims 1 to 13,
A reaction apparatus comprising drive means for moving the cells in the flow path. A reaction apparatus comprising the flow path substrate according to claim 14 or 15,
A reaction apparatus, wherein the cells are moved in a flow path by the micropump. A cell crushing device for crushing cells contained in the solution on the upstream side of the filter,
The cell disruption apparatus comprises:
A cell capturing portion provided on the flow path substrate and capturing the cells on the upstream side of the filter;
The reaction apparatus according to claim 16 or 17, further comprising: a crushing voltage applying unit configured to apply a voltage capable of crushing the cell to the cell captured by the cell capturing unit.   14. The flow path substrate according to claim 1, wherein the flow path substrate is used by being attached to a centrifuge for moving a solution or a cell in the solution in the flow path of the flow path substrate.   The centrifuge includes a rotating disk that attaches the flow path substrate containing the solution and rotates around a predetermined rotation axis, and a microscope for visually observing the state of cells in the solution in the flow path substrate. And a visible centrifuge that separates or synthesizes predetermined cells from the solution by applying a centrifugal force to the solution in the channel substrate or the cells in the solution. Item 22. The flow path substrate according to Item 21.

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