JP5390366B2 - Sample manipulation element - Google Patents

Description

  The present invention relates to a sample operation element for performing a sealing operation in a minute space of a sample to be operated or a moving operation in a minute channel.

  For the purpose of easy handling of various trace liquids or measurement of reaction processes thereof, a micro-molded operation element for operating a trace liquid sample has been proposed. As one of such elements, a sheet member made of silicone rubber called PDMS (Polydimethylsiloxane) is used, and a sheet member having a concave or groove-like structure formed on the lower surface side is brought into close contact with the substrate. There are elements that constitute a minute sealing structure, a flow path structure, and the like (see Patent Documents 1 and 2 and Non-Patent Documents 1 to 3 for sample manipulation elements).

  In such an element, a mold is manufactured using a mask on which a pattern corresponding to a minute operation structure for operating a sample is formed, and a PDMS sheet member is manufactured using the mold. This method has advantages such that the mask can be used repeatedly, and that the mold manufactured by the mask can withstand the fabrication of the micro structural element several times to 10 times while maintaining the accuracy. That is, according to the sample operation element having the above configuration, once a mask and a mold are made, the same element can be repeatedly produced by a simple operation and used for the operation of the sample. In addition, the sheet member of PDMS has high adsorptivity to a substrate such as a glass substrate, and is suitable for creation of a microstructure by bringing the substrate and the sheet member into close contact with each other. Further, such an element can be similarly configured even when a sheet member made of a material other than PDMS is used.

JP 2004-33919 A JP 2004-309405 A

Y. Rondelez et al., "Highly coupled ATP synthesis by F1-ATPase single molecules", NatureVol.433 (2005) pp.773-777 Y. Rondelez et al., "Microfabricated arrays of femtoliter chambers allow single moleculeenzymology", Nature Biotechnology Vol.23 (2005) pp.361-365 D. C. Duffy et al., "Rapid Prototyping of Microfluidic Systems in Poly (dimethylsiloxane)", Anal. Chem. Vol.70 (1998) pp.4974-4984

  In recent years, in the sample manipulation element using the substrate and the sheet member as described above, further miniaturization and higher functionality of the structure have been promoted. On the other hand, when the element is miniaturized or the structure thereof is complicated as described above, there is a possibility that it is difficult to ensure sufficient adhesion between the substrate and the sheet member. On the other hand, a method of increasing the adhesion by pressing the sheet member against the substrate is conceivable. However, in such a method, for example, in consideration of the possibility of affecting the sample to be manipulated, it is necessary to carefully adhere the substrate and the sheet member.

  The present invention has been made to solve the above-described problems, and is a sample operation element capable of improving the adhesion between a substrate and a sheet member for forming a microstructure used for sample operation. The purpose is to provide.

In order to achieve such an object, a sample manipulation element according to the present invention includes (1) a lower substrate on which a sample to be manipulated is placed on a placement surface, and (2) a lower surface on the lower substrate. And (3) the upper sheet member operates the sample on the lower surface side of the upper sheet member with the lower substrate placement surface. A gas interposed between the lower substrate and the upper sheet member when the lower surface of the upper sheet member is brought into close contact with the mounting surface of the first concave structure portion for forming the operation structure and the lower substrate have a second concave structure portion for forming a discharge structure of still escape of liquid, the first concave structure is constituted by a plurality of sealing portions formed in a predetermined two-dimensional array, the The operation structure formed by each sealing part of the concave structure part is It is a concave sealing structure that performs a sealing operation in a fixed space, and the second concave structure part is a discharge structure part that is a two-dimensional combination of hexagonal passage structure parts that connect the sealing parts with straight lines. is constituted by a discharge structure formed by the channel structure of the second concave structure is characterized by groove-like channel structure der Rukoto.

  In the sample manipulation element described above, the manipulation element is configured by arranging an upper sheet member made of a predetermined material having adhesion to the substrate on the mounting surface of the lower substrate. As a micro structure formed on the lower surface side of the sheet member, in addition to the first concave structure portion serving as the sample operation structure, a second concave structure portion functioning as a gas or liquid escape passage discharging structure is provided. . In this way, by providing a discharge structure separately from the operation structure between the substrate and the sheet member, gas or liquid that hinders the substrate and the sheet member from being brought into close contact with each other through the discharge structure. As a result, it is possible to improve the adhesion between the substrate and the sheet member for forming the microstructure used for the operation of the sample, and to realize the rapid completion of the adhesion.

  Here, the discharge structure formed by the second concave structure portion between the substrate and the sheet member is a groove-like passage structure having a width of 1 mm or less extending from a predetermined side surface of the upper sheet member to the same or another side surface, or It is preferable to include at least one of a groove-shaped passage structure having a width of 1 mm or less extending from a predetermined side surface of the upper sheet member to a predetermined position inside. According to such a configuration, when the substrate and the sheet member are brought into close contact with each other, gas or liquid can be suitably released to the outside by the discharge passage structure.

  Further, the operation structure formed by the first concave structure portion between the substrate and the sheet member is a concave sealing structure that performs a sealing operation in a space having a maximum width of 1 mm or less with respect to the sample, or the sample. It is preferable to include at least one of a groove-like channel structure that performs a moving operation in a channel having a width of 1 mm or less. Specifically, the operation structure including such a micro-sealed space structure or a micro-channel structure can be appropriately set according to the operation content of the target sample in the element.

  Regarding the material of the sheet member that forms the upper part of the element in close contact with the mounting surface of the substrate, the material that forms the upper sheet member is preferably silicone. In particular, the material constituting the upper sheet member is preferably PDMS. By using such a material, the adhesion between the substrate and the sheet member can be obtained sufficiently and quickly.

  According to the sample operation element of the present invention, the upper sheet member is arranged so as to be in close contact with the mounting surface of the lower substrate to constitute the element, and the sample operation is performed as a microstructure formed on the lower surface side of the sheet member. In addition to the first concave structure portion that becomes the structure, a second concave structure portion that becomes a gas or liquid escape passage discharge structure is provided, thereby forming a micro structure used for sample operation, and a sheet member. It is possible to improve the adhesiveness of the adhesive and to achieve a quick completion of the adhesion when they are brought into close contact.

It is a schematic diagram which shows the preparation methods of the sample operation element which consists of a board | substrate and a sheet | seat member. It is a schematic diagram which shows the preparation methods of the sample operation element which consists of a board | substrate and a sheet | seat member. It is a figure which shows the usage example of the sample operation element which has a micro sealing structure. It is a figure which shows the structural example of the sample operation element which has a microchannel structure. It is a figure shown about the sealing state of the sample in a sample operation element. It is a top view which shows the structure of 1st Embodiment of a sample operation element. It is a top view which shows the structure of 2nd Embodiment of a sample operation element. It is a top view which shows the structure of 3rd Embodiment of a sample operation element. It is a top view which shows the structure of 3rd Embodiment of a sample operation element. It is a top view which shows the structure of 4th Embodiment of a sample operation element. It is a top view which shows the structure of 4th Embodiment of a sample operation element. It is a top view which shows the structure of 5th Embodiment of a sample operation element. It is a perspective view which shows the structure of 6th Embodiment of a sample operation element.

  Hereinafter, preferred embodiments of a sample manipulation element according to the present invention will be described in detail with reference to the drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the dimensional ratios in the drawings do not necessarily match those described.

  A sample operation element according to the present invention is an element having an operation structure such as a minute sealing structure or a channel structure for a sample, and a lower substrate on which a sample to be operated is placed on a placement surface, and a lower substrate And an upper sheet member disposed above. Further, a micro structure such as a concave shape or a groove shape is formed on the lower surface of the upper sheet member, and an operation structure for the sample is configured by the close contact between the mounting surface of the substrate and the lower surface of the sheet member. First, a manufacturing method, a basic configuration, and a usage pattern of such a sample manipulation element will be described.

  Here, in the following, a case where PDMS (Polydimethylsiloxane) is mainly used as an example of the material constituting the sheet member above the element will be described as an example. The micro structure of PDMS using MEMS technology (microchannels, etc.) is expected to develop industrially due to the small amount of sample, the miniaturization in combination with other devices, and the low price. Some are already in practical use.

  PDMS can transfer up to sub-micron structure by molding, self-adsorbing, can be sealed just by sticking to the substrate, colorless and transparent, almost no autofluorescence, biological sample There are advantages such as not adversely affecting the above. In the bio / chemical field, there has been an attempt to develop a sample manipulation element using PDMS by providing a function to the microchannel itself. Examples are the arrangement of a three-dimensional flow path incorporating a micro pump and the enhancement of functionality by adding a partial enzyme.

  FIG. 1 and FIG. 2 are schematic views showing an example of a method for producing a sample manipulating element including the lower substrate and the upper sheet member. In this method, first, as shown in FIG. 1A, a substrate 80 used for a mold for producing a sheet member is prepared, and a photocurable resin 82 such as SU-8 is formed on the upper surface thereof by a spin coater. Apply.

  Next, as shown in FIG. 1B, using a mask 84 in which a mask pattern 86 is formed on a substrate 85, the photocurable resin 82 on the substrate 80 is irradiated with light from, for example, a mercury lamp. Then, the pattern is exposed. At this time, the corresponding portion of the photocurable resin 82 is cured for the light transmitting portion of the mask 84 where the mask pattern 86 is not formed. As the substrate 85 of the mask 84, for example, a substrate such as BK7, soda glass, quartz or the like having high light transmittance is used. Such a mask substrate is preferably selected in consideration of a target spatial resolution in device fabrication. For the mask pattern 86, for example, a metal such as chromium is used.

  After the exposure of the pattern, as shown in FIG. 1 (c), the substrate 80 and the photocurable resin 82 are heated to accelerate the curing, and further, development and washing are performed as shown in FIG. 1 (d). Then, the uncured portion of the photo-curable resin 82 is removed. As a result, a mold is completed in which the convex portions 83 obtained by curing the photocurable resin on the substrate 80 are formed in a predetermined pattern (a pattern in which the pattern on the mask 84 is transferred).

  Subsequently, an upper sheet member is produced using a mold including the substrate 80 and the convex portion 83 described above. Here, as shown in FIG. 2A, PDMS, which is a thermosetting resin, is applied onto the substrate 80 on which the convex portions 83 are formed, and the PDMS is heated and cured. Thereby, the upper sheet member 20 by PDMS is formed. Further, in the sheet member 20, the concave structure portion 22 is formed in a portion corresponding to the convex portion 83 of the mold on the lower surface side.

  When the sheet member 20 having the concave structure 22 is completed, after cooling, the upper sheet member 20 in which PDMS is cured is peeled off from the mold as shown in FIG. On the other hand, separately from the sheet member 20, a lower substrate 10 whose upper surface 12 serves as a mounting surface for a sample to be operated is prepared. And as shown in FIG.2 (c), it is set as the sample operation element by mounting the sheet | seat member 20 on this board | substrate 10. FIG. The upper sheet member 20 is disposed such that the lower surface thereof is in close contact with the placement surface 12 of the lower substrate 10. At this time, the operation structure 23 for operating the sample is placed between the mounting surface 12 of the substrate 10 and the lower surface of the sheet member 20 by the concave structure portion 22 formed on the lower surface side of the sheet member 20. It is formed. As the lower substrate 10, for example, an observation glass substrate (slide glass) can be used.

  In such a manufacturing method, the operation structure pattern by the concave structure portion 22 between the substrate 10 and the sheet member 20 can be arbitrarily set by the mask pattern 86 of the mask 84 used for mold manufacture of the sheet member 20. it can. This method is an inexpensive manufacturing technique for a fine structure, and is expected to be developed as a chip for biosensors, for example.

  Here, the depth of the concave structure portion 22 formed in the sheet member 20 is set by the exposure time with the mercury lamp in the process of FIG. Further, the thickness of the sheet member 20 is set by the resin application thickness in the step of FIG. The thickness of such a sheet member 20 may be, for example, about 10 μm to several mm or several cm depending on the specific configuration of the operation element. In addition to the methods shown in FIGS. 1 and 2, for the production of the sheet member, various methods and production apparatuses may be used specifically, for example, when a dedicated apparatus that does not use a mask is used. Is possible.

  As a micro operation structure provided in the sample operation element, specifically, for example, a concave sealing structure (sample) that performs a sealing operation on a sample in a space of a predetermined size (preferably a size having a maximum width of 1 mm or less). Can be used. FIG. 3 is a diagram showing an example of use of a sample manipulation element having a microencapsulation structure.

  In this use example, first, as shown in FIG. 3A, a sample solution 90 prepared with a molecular machine 92 with operating magnetic beads attached thereto is prepared in advance on the mounting surface 12 of the lower substrate 10. Keep it. In this state, as shown in FIG. 3B, the upper sheet member 20 in which a plurality of minute concave structure portions 22 having a circular planar structure are formed on the lower surface side is disposed on the substrate 10. As a result, as shown in FIG. 3C, a microchamber 23 having a sealed structure 22 is formed between the substrate 10 and the sheet member 20, and the molecular machine 92 is placed in the microchamber 23. The containing sample solution 90 is sealed.

  Further, as another example of the micro operation structure provided in the sample operation element, a groove-like channel structure (sample is used for performing a moving operation in a channel having a predetermined width (preferably a width of 1 mm or less) with respect to the sample. A flow channel) can be used. FIG. 4 is a diagram illustrating a configuration example of a sample manipulation element having a microchannel structure.

  In this configuration example, a groove-shaped concave structure portion 24 is formed on the lower surface side of the sheet member 20 disposed on the substrate 10, and the micro-channel which is a flow channel structure is configured by the concave structure portion 24. Yes. The concave structure 24 includes an input channel 241 to which a sample solution is input, a first output channel 242 and a second output from which the sample solution from the input channel 241 branches and is output. And a flow path portion 243. Thereby, the sample operation element of the present configuration example has a sorter type configuration.

  An input port 246 that is a through-hole penetrating the sheet member 20 from the upper surface to the lower surface is provided at the end of the input flow path portion 241, and an input tube 251 for sample solution input is connected to the input port. ing. In addition, a first output port 247 and a second output port 248 each having a through hole are provided at end portions of the output flow path portions 242, 243, and output tubes 252, 253 are connected to these output ports. ing.

  In the above configuration, the tubes 251, 252, and 253 are attached to the input port 246 and the output ports 247 and 248 using, for example, a special jig. Further, in the configuration in which a tube or the like is attached in this way, a thick member of about several mm is preferably used as the sheet member 20. Further, the flow rate of the input solution and the output solution in each channel can be controlled by, for example, a high performance pump. In general, the flow paths from the output flow path portions 242 and 243 are directly guided to the input system of the detector, but when measuring or discriminating the components of the solution in each part of the micro flow path There is also. In the future, such usage of sample manipulation elements is expected to increase.

  As described above, in the sample manipulation element constituted by the substrate 10 and the sheet member 20, it becomes difficult to ensure sufficient adhesion between the substrate and the sheet member as the structure becomes finer and more complicated. The problem arises. FIG. 5 is a diagram showing a sealed state of the sample in the sample operation element having a micro-sealing structure. Here, a sheet member 20 made of PDMS in which a plurality of microchambers having a diameter of 10 μm and a capacity of about 1 pL are formed as a sample operating structure is manufactured by molding by the above method, and the sheet member 20 is used to be prepared on the glass substrate 10. 2 shows an example of sealing a sample solution of a molecular machine.

  5, FIG. 5 (a) shows an optical microscope image of the planar structure of the PDMS sheet member 20 in which a plurality of micro chambers are formed. FIG. 5B shows an enlarged optical microscope image of the microchamber in a state where the sample solution on the substrate 10 is sealed. In FIG. 5B, the black dots are 0.7 μm diameter magnetic beads added for molecular machine operation.

  In this example, it is a PDMS sheet member that is originally highly adsorbable to the substrate, but it can be seen that the solution remains between the substrate and the sheet member at the edge of the microchamber, and that adhesion is not realized. In such a case, a method of mechanically pressing the sheet member from above to remove the solution is conceivable, but in the μm region, the molecular machine in the sample solution becomes a substrate due to the mechanical pressure and the flow of the solution generated thereby. The sample itself may be affected. Further, such a problem of adhesion between the substrate and the sheet member is considered to be remarkable when the sheet member is thinned. Further, such a problem is not limited to the liquid remaining between the substrate and the sheet member as described above, and the same problem may occur when a gas (gas) remains.

  On the other hand, in the processing of sheet members by PDMS, surface treatment such as ozone or plasma treatment, parylene vapor deposition, etc., improves adsorption on glass substrates, adsorption on hydrophobic surfaces, and improves resistance to organic solvents, etc. There is. However, even when these methods are used, when the mounting surface of the lower substrate and the lower surface of the upper sheet member are brought into close contact with each other, a gas or liquid that remains between the substrate and the sheet member and hinders the close contact is left. It is difficult to eliminate completely. Even in such a case, the sample manipulation element according to the present invention realizes good adhesion between the substrate constituting the element and the sheet member.

  FIG. 6 is a plan view showing the configuration of the first embodiment of the sample manipulation element according to the present invention. In each embodiment shown below, among the lower substrate 10 and the upper sheet member constituting the sample manipulation element, the planar structure of the sheet member, in particular, the pattern of the concave structure portion formed on the lower surface side of the sheet member. The configuration of the operation element will be described. The cross-sectional structure of the sheet member in each embodiment, the overall configuration of the sample operation element by the close contact between the sheet member and the substrate 10, the manufacturing method thereof, and the like are the same as those described with reference to FIGS.

  6A shows the pattern structure of the first concave structure portion 31 among the concave structure portions of the sheet member 30 according to the present embodiment, and FIG. 6B shows the second concave structure portion 33. FIG. 6C shows the entire concave structure pattern on the lower surface side of the sheet member 30 including the first and second concave structure portions 31 and 33. Further, in the present embodiment, an example is shown in which the sample operation structure has a reactor type configuration.

  Among the concave structure portions provided on the lower surface side of the sheet member 30, the first concave structure portion 31 is an operation structure (in FIG. 2C) for operating the sample with the placement surface 12 of the lower substrate 10. For forming the operating structure 23). In the present embodiment, the first concave structure portion 31 has the first input flow channel portion 311 and the second input flow channel portion 312 to which the sample solution is input, and the sample solution from the input flow channel portions 311 and 312 merge. Then, the intermediate flow path portion 313 serving as a reaction path thereof, and the first output flow path portion 314 and the second output flow path portion 315 from which the sample solution from the intermediate flow path portion 313 branches and is output respectively. It is configured.

  A first input port 321 and a second input port 322 each having a through hole penetrating the sheet member 30 from the upper surface to the lower surface are provided at the ends of the input flow channel portions 311 and 312 opposite to the intermediate flow channel portion 313. Is provided. In addition, a first output port 324 and a second output port 325 each including a through hole are provided at the end of the output flow channel portions 314 and 315 opposite to the intermediate flow channel portion 313. The through holes of these input ports 321 and 322 and output ports 324 and 325 are formed in the sheet member 30 by a method such as double exposure or later processing.

  By means of each of these flow path portions formed as the first concave structure portion 31 on the lower surface side of the upper sheet member 30, a micro flow path (preferably a width) is preferably used as a sample operation structure between the lower substrate and the sample. A groove-like channel structure for performing a moving operation in a channel of 1 mm or less is formed. The configuration conditions of such a channel structure are, for example, a channel width of about 100 μm and a diameter of through holes of the input port and the output port of about 5 mm. Specific configuration conditions of the channel structure such as the channel width are appropriately set and changed according to the purpose of each operation element.

  In contrast to the first concave structure portion 31, the second concave structure portion 33 is configured such that when the lower surface of the upper sheet member 30 is brought into close contact with the placement surface 12 of the lower substrate 10, the substrate 10 and the sheet member 30. Is used to additionally form a discharge structure (gas or liquid discharge flow path) serving as a gas or liquid escape path interposed between the first concave structure portion 31 and the first concave structure portion 31. is there. Such a discharge structure by the concave structure portion 33 is preferably constituted by a groove-like passage structure having a predetermined width (preferably a width of 1 mm or less) extending from a predetermined side surface of the sheet member 30 to the same or another side surface. .

  In the present embodiment, the second concave structure portion 33 includes the discharge structure portion 331 formed in the left side of the sheet member 30 in the drawing and the region sandwiched by the flow path portions 311 and 312, the upper side and the flow path of the sheet member 30. A discharge structure portion 332 formed in a region sandwiched by the portions 311, 313, 314; a discharge structure portion 333 formed in a region sandwiched by the lower side of the sheet member 30 and the flow path portions 312, 313, 315; and the sheet member 30 and a discharge structure portion 334 formed in a region sandwiched between the flow path portions 314 and 315.

  Each of these discharge structure portions 331 to 334 is configured by an aggregate of a plurality of groove-shaped passage structures extending from the side surface of the sheet member 30 to the same side surface. About the width | variety of such a channel | path structure, it is good also as the width | variety similar to the flow-path structure by the 1st concave structure part 31, or good also as a different width | variety. When a glass substrate is used as a mask for producing the mold of the sheet member 30, a flow path having a width of about 10 μm can be designed. In addition, when a quartz substrate is used for the mask, a flow path with a width of about 1.5 μm can be designed.

  The effect of the sample manipulation element according to the above embodiment will be described.

  In the sample manipulation element shown in FIG. 6, an upper sheet member 30 made of a predetermined material having adhesion to the lower substrate 10 is arranged on the mounting surface of the substrate 10 to constitute the manipulation element. And as a micro structure formed on the lower surface side of the sheet member 30, in addition to the first concave structure portion 31 that becomes the sample operation structure, a second concave structure portion 33 that functions as a gas or liquid escape passage discharging structure is provided. Provided. In this way, by providing an additional discharge structure separately from the intended operation structure between the substrate 10 and the sheet member 30, a gas or liquid that interferes when the substrate and the sheet member are brought into close contact with each other, It is possible to escape to the outside through this discharge structure, improving the adhesion between the substrate and the sheet member for forming the micro structure used for sample operation, and realizing a quick completion of the adhesion. It becomes.

  The pattern of the discharge structure by the second concave structure portion 33 is arbitrarily set according to the mask pattern in the mask used for mold production of the sheet member 30 together with the pattern of the operation structure by the first concave structure portion 31. be able to. Therefore, it is possible to set the discharge channel pattern suitable for the specific operation structure of each operation element.

  Here, as described above, the discharge structure formed by the second concave structure portion between the substrate and the sheet member has a groove shape with a width of 1 mm or less extending from a predetermined side surface of the sheet member to the same or other side surface. It is preferable to include a passage structure. In general, the discharge structure by the second concave structure portion is a groove-like passage structure having a width of 1 mm or less extending from a predetermined side surface of the sheet member to the same or another side surface, or a predetermined internal portion from a predetermined side surface of the sheet member. It is good also as a structure containing at least one of the groove-shaped channel | path structures of width 1mm or less extended to a position. According to such a configuration, when the lower substrate and the upper sheet member are brought into close contact with each other, excess gas or liquid can be suitably released to the outside by the discharge passage structure.

  Silicone (silicone resin, silicone rubber) is preferably used for the material of the sheet member 30 that is in close contact with the mounting surface 12 of the substrate 10 and constitutes the upper portion of the element. In particular, as the material of the sheet member, it is preferable to use the above-described PDMS that has excellent adhesion to the substrate. By using such a material, the adhesion between the substrate and the sheet member can be secured sufficiently and quickly. In addition to the above, various materials may be used as the material for the sheet member as long as the operation structure and the discharge structure can be formed in close contact with the substrate. Examples of such materials include polydiene polymers, polyvinylidene chloride (PVDC), polyvinylidene fluoride, polyvinylidene halide, synthetic resins such as polyethylene, natural rubber, and the like.

  FIG. 7 is a plan view showing the configuration of the second embodiment of the sample manipulation element. 7A shows the pattern structure of the first concave structure portion 36 among the concave structure portions of the sheet member 35 according to the present embodiment, and FIG. 7B shows the second concave structure portion 37. FIG. 7C shows the entire concave structure pattern on the lower surface side of the sheet member 35 including the first and second concave structure portions 36 and 37. Moreover, in this embodiment, the example which made the operation structure of the sample the structure of reaction tank type (reaction well type) is shown.

  In the present embodiment, the first concave structure portion 36 for the sample manipulation structure is formed by a plurality of sealing portions (concave portions, circular dots) 361 formed in a predetermined two-dimensional array and each having a circular planar shape. It is configured. By these sealing portions 361 on the lower surface side of the upper sheet member 35, the space between the lower substrate and the sample is preferably within a minute space (preferably a space having a maximum width of 1 mm or less, or a space having a diameter of 1 mm or less in the case of a circle). A concave sealing structure for performing a sealing operation is formed. The configuration condition of such a sealing structure is, for example, that the diameter of the circular dot of the concave sealing portion 361 is about 2 μm, the two-dimensional arrangement structure of the dots is a hexagonal lattice arrangement, and the 60 ° direction between the dots The pitch is about 4 μm. The specific configuration conditions of the sealing structure such as the dot diameter are appropriately set and changed according to the purpose of each operation element, as in the case of the flow path structure. For example, generally, the diameter of the sealing portion 361 serving as a microchamber for sealing a sample can be set to about 2 μm to 10 μm.

  In contrast to the first concave structure portion 36 as described above, the second concave structure portion 37 is configured by a discharge structure portion 371 in which hexagonal passage structures in which dots are connected by a straight line are two-dimensionally combined. In this case, the width of the groove-shaped passage structure in the discharge structure portion 371 is preferably set to the same width as the diameter of the concave sealing portion 361. Moreover, in such a discharge | emission structure part 371, as illustrated by the broken line 38 in FIG.7 (c), the edge part of each channel | path structure is exposed to the side surface of a sheet | seat member by cutting out a sheet member, thereby gas Alternatively, a discharge passage serving as a liquid escape path is formed.

  As shown in FIGS. 6 and 7, the operation structure formed by the first concave structure portion between the substrate and the sheet member is sealed in a space having a maximum width of 1 mm or less with respect to the sample. It is preferable to include at least one of a concave sealing structure that performs the above or a groove-shaped channel structure that performs a moving operation in a channel having a width of 1 mm or less with respect to the sample. Specifically, the operation structure including such a micro-sealed space structure or a micro-channel structure can be appropriately set according to the operation content of the target sample in the element.

  In addition, since the sheet member having the sealing structure as shown in FIG. 7 has higher symmetry in the planar structure than the sheet member such as the flow path structure, a large sheet is prepared, and then the sheet member is cut out. May be used. In such a case, the discharge structure portion 371 combined with the hexagonal passage structure as described above sacrifices the number of microchambers for sealing the sample, but the gas or the gas from any side of the cut sheet member. There is an advantage that the liquid can be sufficiently discharged.

  However, also in the sheet member having such a sealing structure, various configurations other than the hexagonal passage structure described above may be used as the gas and liquid discharge structure. For example, the discharge structure may be configured by a linear passage structure provided between the circular dot patterns of the sealing portion 361. In general, a mask pattern for forming a mold for determining the pattern of the first and second concave structure portions on the lower surface side of the sheet member is designed by CAD. For example, when the mask pattern is designed, Alternatively, a passage structure pattern serving as a gas or liquid discharge flow path may be designed, and a sealing structure pattern may be disposed in the gap later.

  Further, as described above with reference to FIG. 3, when a sample solution is prepared in advance on the substrate mounting surface, and then a sheet member is installed on the substrate to seal the sample solution in the microchamber. For example, by applying an appropriate force to the entire surface of the sheet member and using a filter paper having high water absorption at the edge of the sheet member, the solution discharged from the discharge structure is sucked to adhere the substrate and the sheet member. Can be realized more reliably. In the case of discharging the gas between the substrate and the sheet member, for example, the substrate having the sheet member placed on the mounting surface is placed in a vacuum chamber, and the residual gas is obtained by making a certain vacuum state, A method of discharging the residual liquid can be used.

  As an example of the sample manipulation element, the upper sheet member having the configuration shown in FIG. 7 was produced by the method shown in FIGS. 1 and 2, and the sample manipulation element was produced by combining with the lower substrate. In this embodiment, first, a silicon wafer having SU-8 spin-coated on its upper surface is prepared, and a mask having a mask pattern corresponding to the configuration shown in FIG. Pattern exposure was performed. Regarding the setting conditions of the spin coater when applying SU-8, the press pin was held at 1000 rpm for 10 seconds and the top spin was held at 4000 rpm for 30 seconds.

After soft baking (2 minutes on a hot plate at 90 ° C. and 1 minute in an oven at 90 ° C.), the exposure was performed using a general-purpose exposure apparatus with the mask in direct contact with SU-8. At that time, in order to avoid sticking between SU-8 and the mask surface, in addition to the commonly performed pre-baking (90 ° C., 2 minutes), baking was additionally performed in a 95 ° C. oven for 2 minutes. Further, the film was naturally cooled on the wipe for 3 minutes and exposed for 30 seconds. The exposure energy at this time was 300 mJ / cm ² .

  Then, post-exposure heating (PEB) is performed on a hot plate (1 minute at 60 ° C., 3 minutes at 90 ° C.), naturally cooled on a wipe for 3 minutes, and developed in a dedicated developer (SU-8 developer). Performed (primary development for 2 minutes, secondary development for 1 minute). Further, the substrate was rinsed with a 2-propanol solution for 30 seconds, sprayed with nitrogen gas and dried, and incubated in a 90 ° C. oven for 90 minutes to completely cure the SU-8 resin. The wafer cured with SU-8 was stored under clean conditions.

  The PDMS used as the material for the upper sheet member was commercially available from Corning. 1/10 volume of the polymerization initiator was added to the resin solution immediately before the operation, and after stirring, it was deaerated with an aspirator in a vacuum disk for about 10 minutes. And PDMS was dripped on the silicon wafer in which the mold pattern by SU-8 arranged in the plastic petri dish was formed, and the whole petri dish was rotated (turned so that it might spread), and it extended thinly. Then, again, the petri dish was deaerated with an aspirator in a vacuum disk, and left in a 90 ° C. oven for 90 minutes to cure PDMS. After cooling, in the part where the sealing structure pattern and discharge structure pattern of the micro chamber are created, the area to be used is cut out from the wafer with a cutter and peeled off using tweezers to form the upper sheet member, which is clean until use Stored under mild conditions.

As a sample, a molecular machine (F ₁ -ATPase) was used. This molecular machine has three common subunits, and ten His-Tags are extended from the N-terminal of the subunits by genetic engineering. Molecular machines can be aligned and aligned on a Ni-NTA modified glass substrate. This molecular machine has been found to be a rotating molecular motor that consumes ATP and rotates. By changing two residues of the rotor subunit to Cys and reacting with maleimide biotin, a streptavidin-modified bead is produced. Designed to adsorb specifically.

By a general method, F ₁ -ATPase was placed on the glass substrate so that the directions thereof were aligned, and beads (˜1 μm) were attached to the rotor as a mark of rotation. All these operations are performed in solution. About 20 μL of the solution is left, and F ₁ -ATPase and beads that could not be adsorbed are removed by repeatedly exchanging the solution. The final solution contains 2 mM MgATP.

  Observation was started by gently placing a PDMS sheet member on the sample solution prepared on the glass substrate. If the thickness of the PDMS sheet is reduced to some extent, the operation of placing the sheet member uniformly on the solution becomes difficult. After the sheet member is placed, the solution that oozes out on the side of the sheet is sucked out by a piece of filter paper. As described above, in the sheet member provided with the discharge structure pattern serving as a gas or liquid escape path in addition to the sealing structure pattern, the excess solution smoothly oozes out, so that the gas and liquid remain in the sheet. There was no case.

  In the microscopic observation of the substrate in which the sheet member having the above configuration was used and the solution was sufficiently sucked out from the edge by the filter paper, it was confirmed by an optical microscope that the region of each microchamber was completely sealed. Here, the free beads in the chamber that could not be removed due to the exchange of the solution had Brownian motion, and the beads outside the chamber (edge) were easily confirmed by being pressed against the PDMS and not moving. be able to.

  In addition, about the sheet | seat member which consists of PDMS, effects, such as an improvement of the tolerance to an organic solvent, can be acquired by surface treatments, such as parylene vapor deposition, as mentioned above. Parylene is a common name for polyparaxylylene. In general, since the surface of the material to be coated is coated by polymerization of a reactive gas, a conformal coating without a pinhole can be realized.

  Coated parylene has high insulation properties, excellent mechanical properties, and is stable both optically and chemically. To improve the reliability of various IC and other machines. It is used. In some cases, the surface of the PDMS sheet or the surface of the substrate is used to increase the insulation and hydrophobicity. The use of a hydrophobic surface can avoid nonspecific adsorption to a flow path or a reaction tank, for example, when a very small amount of enzyme solution is used.

  The configuration of the sample manipulation element according to the present invention will be further described.

  8 and 9 are plan views showing the configuration of the third embodiment of the sample manipulation element. 8A shows the pattern structure of the first concave structure portion 41 among the concave structure portions of the sheet member 40 according to the present embodiment, and FIG. 8B shows the second concave structure portion 43. The pattern structure is shown. FIG. 9 shows the entire concave structure pattern on the lower surface side of the sheet member 40 including the first and second concave structure portions 41 and 43. Further, in the present embodiment, an example is shown in which the sample operation structure has a reactor type configuration. The reactor-type sample manipulation element has at least one reaction tank for a plurality of types of solutions in a micro-channel of a sample solution, and is intended for mixing a large number of types of solutions in the reaction tank and achieving subsequent reactions. Is.

  In the present embodiment, the first concave structure portion 41 for the sample operation structure includes a first input channel portion 411 and a second input channel portion 412 to which a sample solution is input, an input channel portion 411, And an output channel portion 413 to which the sample solutions from 412 are joined and output.

  A first input port 421 and a second input port 422 each including a through hole are provided at the end of the input flow channel portions 411 and 412 on the opposite side to the output flow channel portion 413. Further, an output port 423 formed of a through hole is provided at the end of the output flow channel portion 413 opposite to the input flow channel portions 411 and 412. Further, mixing and reaction of the solutions from the input flow path portions 411 and 412 occur between the input flow path portions 411 and 412 and the output flow path portion 413, and the solution after the reaction is supplied to the output flow path portion 413. A reaction tank (reaction chamber) 425 is provided.

  With respect to the first concave structure portion 41, the second concave structure portion 43 for the discharge structure serving as a gas or liquid escape path between the substrate and the sheet member 40 has a left side and a flow direction of the sheet member 40. It is formed in a region sandwiched by the path portions 411 and 412 and mainly in a region sandwiched by the discharge structure portion 431 that serves as an escape path to the left side of the sheet member 40 and the upper and right sides of the sheet member 30 and the flow path portions 411 and 413. The sheet member 40 is mainly formed in a region sandwiched by the discharge structure portion 432 that serves as an escape path to the upper side and the right side of the sheet member 40 and the lower side, the right side, and the flow path portions 412 and 413 of the sheet member 30. And a discharge structure portion 433 serving as an escape path to the lower side and the right side. These discharge structure portions 431 to 433 are each configured as an aggregate of a plurality of groove-shaped passage structures, and are formed in a passage structure pattern in which gas or liquid is uniformly discharged in each direction. .

  FIG. 9 shows the size of each part of an example in which the sample manipulation element according to the present embodiment is specifically configured. The unit of each size shown in the figure is μm. In the following, when the distance between the flow paths and the passages is indicated, the distance between the centers of the flow paths is shown in principle. Moreover, the width | variety (width | variety of a discharge flow path) of the groove-shaped channel | path structure which comprises the 2nd concave structure part 43 is 100 micrometers, for example.

  Here, in general, in the configuration of a sample operation element having an operation structure for operating a sample and a discharge passage structure serving as a gas and liquid escape path, each sealing structure, flow path structure, and escape path in the operation structure The sheet member can be bonded if the distance to the passage structure is approximately the same as the width of the flow path structure or the width of the passage structure of the escape passage. Further, as the sealing structure and the flow path structure become finer, it is preferable to form the passage structure of the escape passage closer to the operation structure. At this time, the distance between the two may be about the width of the passage structure of the escape path.

  10 and 11 are plan views showing the configuration of the fourth embodiment of the sample manipulation element. 10A shows the pattern structure of the first concave structure portion 51 among the concave structure portions of the sheet member 50 according to the present embodiment, and FIG. 10B shows the second concave structure portion 53. The pattern structure is shown. FIG. 11 shows the entire concave structure pattern on the lower surface side of the sheet member 50 including the first and second concave structure portions 51 and 53. Further, in the present embodiment, an example is shown in which the sample operating structure has a sorter type configuration. The sorter type sample manipulation element is intended to discriminate a target substance contained in an input sample solution.

  In the present embodiment, the first concave structure portion 51 for the sample operation structure is used for the input flow path portion 511 to which the sample solution is input and the sheath flow (support flow that flows outside so as to wrap the sample solution). Introducing flow channel portions 512 and 513 for introducing the solution, and an intermediate flow channel portion in which the sample solution from the input flow channel portion 511 and the sheath flow solution from the introducing flow channel portions 512 and 513 merge to flow in a laminar flow state. 514, and a first output flow path section 515 and a second output flow path section 516 to which the solution from the intermediate flow path section 514 is branched and output, respectively. These output flow path portions are configured, for example, as a target solution output flow path for target substance output, which is distinguished from the first output flow path section 515, and a second output flow path section 516 as a waste liquid output flow path for waste liquid output. Is done.

  An input port 521 formed of a through hole is provided at the end of the input flow path portion 511 opposite to the intermediate flow path portion 514. In addition, introduction ports 522 and 523 each having a through hole are provided at the ends of the introduction flow channel portions 512 and 513 opposite to the intermediate flow channel portion 514. Further, at the end of the output flow path portions 515 and 516 opposite to the intermediate flow path portion 514, a first output port (target solution output port) 525 made of a through hole and a second output port (waste liquid output port), respectively. ) 526 is provided.

  As the target substance contained in the sample solution, for example, a substance obtained by specifically adsorbing and binding a fluorescent dye to target cells or beads by a fluorescent antibody method is used. Further, the intermediate flow path part 514 is formed so that the flow path width gradually becomes narrower from the input flow path part 511 side toward the output flow path part 515, 516 side, and a part thereof is the target substance. The detection unit 517 is used for detection. The detection of the target substance by the detection unit 517 is performed, for example, by detecting light emission from the fluorescent dye in the target substance as described above. Further, the branching portion from the intermediate flow path portion 514 to the output flow path portions 515 and 516 serves as a discrimination section 518 used for discriminating between the sample solution containing the target substance and the waste liquid not containing the target substance. The discrimination of the target substance by the discrimination unit 518 is performed by changing the flow path of the target substance by, for example, a magnetic field, an electric field, or light radiation.

  The second concave structure portion 53 for the discharge structure serving as a gas or liquid escape path between the substrate and the sheet member 50 with respect to the first concave structure portion 51 is provided with the input flow path portion 511 and the introduction. The sheet is formed in a region on the left side of the flow channel portions 512 and 513 and formed in a region above the discharge structure portion 531 serving as a escape path to the left side of the sheet member 50, the intermediate flow channel portion 514 and the output flow channel portion 515, A discharge structure portion 532 serving as an escape path to the upper side of the member 50, a discharge structure portion 533 formed in a region below the intermediate flow path portion 514 and the output flow path portion 516, and serving as a escape path to the lower side of the sheet member 50; A discharge structure portion 534 that is formed in the right region of the output flow path portions 515 and 516 and serves as an escape path to the right side of the sheet member 50. These discharge structure portions 531 to 534 are each configured as an aggregate of a plurality of groove-shaped passage structures.

  In addition, in FIG. 11, the size (micrometer) of each part is shown about an example at the time of specifically configuring the sample operation element by this embodiment. Among the numerical values of the sizes shown in the figure, the distance 180 μm between the output flow path part and the outer discharge structure part, and the distance 440 μm between the output flow path part and the inner discharge structure part, It is not the distance between the centers of the roads, but the distance between the edges of the flow paths.

  The width of each flow path in the first half of the input flow path part 511, the introduction flow path parts 512, 513, and the intermediate flow path part 514 is, for example, 200 μm. Moreover, the width | variety of each flow path of the latter half part of the intermediate flow path part 514 and the output flow path parts 515 and 516 is 40 micrometers, for example. Moreover, the diameter of each through-hole of the input port 521, the inlet ports 522 and 523, and the output ports 525 and 526 is 2000 micrometers, for example. Further, the width of the groove-shaped passage structure constituting the second concave structure portion 53 is, for example, 100 μm.

  FIG. 12 is a plan view showing the configuration of the fifth embodiment of the sample manipulation element. 12, FIG. 12 (a) shows the overall structure of the concave structure portion of the sheet member 60 according to the present embodiment, and FIG. 12 (b) is a partially enlarged view of the structure shown in FIG. 12 (a). FIG. 12 (c) shows a further enlarged view of the structure shown in FIG. 12 (b). Further, in the present embodiment, an example in which the operation structure of the sample is configured as a trap type is shown. The trap-type sample manipulation element is a micro-channel element that is devised for fixing a target substance such as a bead or a cell to a specific site in an observation field.

  In the present embodiment, the first concave structure 61 for the sample operation structure has a trap flow provided between the left input port 612 for inputting the sample solution and the right output port 613 for outputting the solution. It is comprised by the road part 611. The trap channel section 611 is divided into three stages of channel sections in the vertical direction, as schematically shown in FIG. The trap channel section is configured to flow the sample solution from the input port 612 from left to right while meandering up and down. The middle trap channel section is configured to flow the sample solution from the upper trap channel section from right to left while meandering up and down. Further, the lower trap channel section is configured to flow the sample solution from the middle trap channel section from the left to the right while meandering up and down and to output to the output port 613.

  As shown in the enlarged views of FIGS. 12B and 12C, each of the flow paths 616 meandering up and down in the trap flow path section 611 is connected with a shortcut between the meandering flow paths 616. A plurality of trap channels 617 configured in such a way that the target substance in the sample solution is trapped in the middle are arranged along the channels. In such a configuration, the target substance in the sample solution is trapped in order from the upstream trap channel 617 in each of the plurality of trap channels 617. For example, 3000 trap channels 617 are arranged in each of the upper, middle, and lower trap channel sections.

  With respect to the first concave structure portion 61, the second concave structure portion 63 for the discharge structure serving as a gas or liquid escape path between the substrate and the sheet member 60 includes a trap channel portion 611, an input. It is formed with respect to the entire surrounding area excluding the area where the port 612 and the output port 613 are provided. The concave structure portion 63 can discharge residual gas and liquid left and right and up and down at the four corners of the sheet member 60 and can be discharged left and right near the trap channel portion 611 by an aggregate of a plurality of groove-shaped passage structures. It is configured.

  In FIG. 12A, the size (μm) of each part is shown for an example in which the sample manipulation element according to the present embodiment is specifically configured. The size of the groove-shaped passage structure constituting the second concave structure portion 63 is, for example, a region in the vicinity of the trap channel portion 611, a width of 40 μm, a pitch of 100 μm, and an outer region (upper and lower in the drawing). The width is about 100 μm and the pitch is about 1000 μm.

  FIG. 13 is a diagram showing the configuration of the sixth embodiment of the sample manipulation element. In FIG. 13, the second concave structure portion for the discharge structure that serves as a gas or liquid escape path is not shown. Moreover, about the 1st concave structure part for the operation structure of a sample, the structure is simplified and shown typically.

  In the present embodiment, the first sheet member 70 in which the first concave structure portion 72 serving as a flow channel structure is formed and the first concave structure portion 77 also serving as a flow channel structure are formed on the lower substrate 10. Two sheet members of the second sheet member 75 are stacked, thereby realizing a three-dimensional flow path structure as a sample operation structure. Further, the sheet members 70 and 75 are additionally provided with second concave structures that serve as gas and liquid discharge structures, respectively.

  Thus, in the sample operation element having a configuration in which two or more sheet members are stacked to achieve high functionality, it is necessary to form each sheet member thinly. For example, in many cases, the current sheet has a thickness of about sub-millimeters, but when a laminated structure is used, it may be necessary to form a structure on a film of about several tens of μm.

  When the sheet member becomes thin in this way, the rigidity of the sheet member itself is lost, and thus the problem of discharge of residual gas and liquid at the time of bonding the substrate and the sheet member becomes more serious. On the other hand, as described above, a plurality of sheet members are stacked in this manner by applying a configuration in which a discharge structure serving as a gas or liquid escape path is formed on the sheet member in addition to the sample operation structure. Also in the configuration, the residual gas and the liquid can be suitably discharged, and the substrate and the plurality of sheet members can be suitably adhered and stacked.

  In the configuration in which a plurality of sheet members are stacked as described above, the alignment between the sheet members is performed, for example, under a microscope. In the configuration example shown in FIG. 13, in the flow path structure 72 of the lower sheet member 70, three flow paths extending in the lateral direction are formed, and reaction tanks are provided at predetermined positions on the flow paths. Yes. Further, in the flow path structure 77 of the upper sheet member 75, three flow paths extending in the vertical direction are formed, and reaction tanks are respectively provided at predetermined positions on the flow path. In such a configuration, the position of the three reaction vessels in each of the sheet members 70 and 75 can be adjusted between the sheet members 70 and 75.

  The sample manipulation element according to the present invention is not limited to the above-described embodiments and configuration examples, and various modifications are possible. For example, the operation structure for operating the sample and the first concave structure portion corresponding to the sheet member should be set appropriately according to the type of sample and the content of the specific operation on the sample. Is possible. Further, the gas or liquid escape structure discharge structure and the corresponding discharge structure pattern of the second concave structure portion can be set as appropriate in accordance with the sealing structure pattern and the flow path structure pattern in the operation structure. preferable.

  INDUSTRIAL APPLICABILITY The present invention can be used as a sample operation element that can improve the adhesion between a substrate and a sheet member for forming a microstructure used for sample operation.

DESCRIPTION OF SYMBOLS 10 ... Lower board | substrate, 12 ... Mounting surface, 20 ... Upper sheet member, 22, 24 ... Concave structure part, 23 ... Operation structure,
30, 35, 40, 50, 60, 70, 75 ... upper sheet member, 31, 36, 41, 51, 61, 72, 77 ... first concave structure (operation structure), 33, 37, 43, 53, 63 ... 2nd concave structure part (discharge structure),
DESCRIPTION OF SYMBOLS 80 ... Board | substrate, 82 ... Photocurable resin, 83 ... Convex part, 84 ... Mask, 85 ... Board | substrate, 86 ... Mask pattern, 90 ... Sample solution, 92 ... Molecular machine.

Claims (5)

A lower substrate on which a sample to be operated is placed on a placement surface;
An upper sheet member disposed on the lower substrate so that a lower surface thereof is in close contact with the mounting surface of the lower substrate;
The upper sheet member is on the lower surface side,
A first concave structure portion for forming an operation structure for operating the sample with the mounting surface of the lower substrate;
When the lower surface of the upper sheet member is brought into close contact with the mounting surface of the lower substrate, a discharge structure serving as a gas or liquid escape path interposed between the lower substrate and the upper sheet member is formed. have a second concave structure for,
The first concave structure portion is constituted by a plurality of sealing portions formed in a predetermined two-dimensional array, and the operation structure formed by each sealing portion of the first concave structure portion is the sample. Is a concave sealing structure that performs a sealing operation in a predetermined space,
The second concave structure portion is constituted by a discharge structure portion that is a two-dimensional combination of hexagonal passage structure portions in which sealing portions are connected by a straight line, and each passage structure portion of the second concave structure portion. It said discharge structure formed by the sample handling device characterized grooved channel structure der Rukoto. Said discharge structure formed by the second concave structure, the same or other width 1mm or less of the groove-like channel structure extending to the side surface from a predetermined side surface of the upper sheet member or a given of said upper sheet member, sample handling device according to claim 1, characterized in that it comprises at least one of the interior of the width 1mm or less and extending to a predetermined position groove-like channel structure from the side. The operating structure formed by the first concave structure section, according to claim 1, wherein the a sealing structure of the concave performing sealing operation within the following space maximum width 1mm on the sample or 2. The sample manipulation element according to 2.   The material which comprises the said upper sheet | seat member is silicone, The sample operation element as described in any one of Claims 1-3 characterized by the above-mentioned.   5. The sample manipulating element according to claim 4, wherein the material constituting the upper sheet member is PDMS.