WO2013031912A1 - Substrat et procédé d'analyse - Google Patents

Substrat et procédé d'analyse Download PDF

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Publication number
WO2013031912A1
WO2013031912A1 PCT/JP2012/072051 JP2012072051W WO2013031912A1 WO 2013031912 A1 WO2013031912 A1 WO 2013031912A1 JP 2012072051 W JP2012072051 W JP 2012072051W WO 2013031912 A1 WO2013031912 A1 WO 2013031912A1
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Prior art keywords
hole
substrate
polymer
molecule
dna
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PCT/JP2012/072051
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English (en)
Japanese (ja)
Inventor
理 額賀
山本　敏
和仁 田端
正和 杉山
Original Assignee
株式会社フジクラ
技術研究組合Ｂｅａｎｓ研究所
国立大学法人東京大学
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Priority to JP2013531404A priority Critical patent/JP6134645B2/ja
Publication of WO2013031912A1 publication Critical patent/WO2013031912A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K26/00—Working by laser beam, e.g. welding, cutting or boring
    • B23K26/02—Positioning or observing the workpiece, e.g. with respect to the point of impact; Aligning, aiming or focusing the laser beam
    • B23K26/06—Shaping the laser beam, e.g. by masks or multi-focusing
    • B23K26/062—Shaping the laser beam, e.g. by masks or multi-focusing by direct control of the laser beam
    • B23K26/0622—Shaping the laser beam, e.g. by masks or multi-focusing by direct control of the laser beam by shaping pulses
    • B23K26/0624—Shaping the laser beam, e.g. by masks or multi-focusing by direct control of the laser beam by shaping pulses using ultrashort pulses, i.e. pulses of 1ns or less
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K26/00—Working by laser beam, e.g. welding, cutting or boring
    • B23K26/0006—Working by laser beam, e.g. welding, cutting or boring taking account of the properties of the material involved
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K26/00—Working by laser beam, e.g. welding, cutting or boring
    • B23K26/36—Removing material
    • B23K26/38—Removing material by boring or cutting
    • B23K26/382—Removing material by boring or cutting by boring
    • B23K26/389—Removing material by boring or cutting by boring of fluid openings, e.g. nozzles, jets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K26/00—Working by laser beam, e.g. welding, cutting or boring
    • B23K26/36—Removing material
    • B23K26/40—Removing material taking account of the properties of the material involved
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K26/00—Working by laser beam, e.g. welding, cutting or boring
    • B23K26/50—Working by transmitting the laser beam through or within the workpiece
    • B23K26/55—Working by transmitting the laser beam through or within the workpiece for creating voids inside the workpiece, e.g. for forming flow passages or flow patterns
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K2103/00—Materials to be soldered, welded or cut
    • B23K2103/30—Organic material
    • B23K2103/42—Plastics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K2103/00—Materials to be soldered, welded or cut
    • B23K2103/50—Inorganic material, e.g. metals, not provided for in B23K2103/02 – B23K2103/26
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K2103/00—Materials to be soldered, welded or cut
    • B23K2103/50—Inorganic material, e.g. metals, not provided for in B23K2103/02 – B23K2103/26
    • B23K2103/56—Inorganic material, e.g. metals, not provided for in B23K2103/02 – B23K2103/26 semiconducting

Definitions

  • the present invention relates to a substrate having a through hole and an analysis method using the substrate. More specifically, the present invention relates to a substrate having a through-hole in which a polymer molecule can be extended and arranged, and a method for analyzing the arrangement of structural units of the polymer molecule using the substrate.
  • DNA sequencers DNA sequencers
  • Patent Document 1 discloses a technique in which a nucleic acid molecule such as DNA extracted from a specimen is handled as a single DNA molecule without being amplified by PCR or the like, and its base sequence is analyzed.
  • DNA strands are easily cleaved by a physical shearing force.
  • the DNA strand to be analyzed is donated to the immobilized DNA synthase in a state in which it is dissolved in the solution. Therefore, the DNA strand is unstable, and due to the shear force generated in the solution, the DNA strand is There is a risk of being cut. For this reason, it is considered that the length of a DNA strand that can be reliably read remains at about 600 to 1000 base pairs as before.
  • the present invention has been made in view of the above circumstances, and a substrate having a through-hole capable of stably arranging a polymer molecule such as a DNA chain to be analyzed, and the polymer using the substrate It is an object of the present invention to provide a method for analyzing the arrangement of molecular constituent units.
  • the substrate according to the first aspect of the present invention includes a base material provided with a space into which a solution containing polymer-like molecules is allowed to flow therein, and is formed inside the base material, and is open to the space.
  • a through-hole having a shape capable of extending and arranging the polymer-like molecule therein, and at least a portion of the base material constituting the through-hole is formed of a single member.
  • the polymer molecule can be kept stable by extending and arranging at least a part of the polymer molecule in the through hole. This is because a turbulent flow with a large shearing force that cuts the polymer-like molecule hardly occurs within the through-hole. Moreover, since the site
  • the strength refers to heat resistance, cold resistance, pressure resistance, chemical resistance, and deformation resistance.
  • part which comprises the said through-hole is formed with the single member, and there is no location and seam bonded together in the said through-hole, it generate
  • the optical signal is refracted and becomes stray light. For this reason, it is easy to optically observe the inside of the through hole.
  • At least a part of the through hole is columnar.
  • the direction in which the polymer molecules extend can be arranged along the longitudinal direction of the columnar through hole. For this reason, it becomes easier to control the arrangement of the polymer-like molecules.
  • the length of the through hole in the longitudinal direction is preferably 0.1 ⁇ m to 10 mm.
  • the length of the through hole in the longitudinal direction is within the above range, it becomes easier to introduce the polymer molecule into the through hole.
  • the through-hole is relatively short, it becomes easier to transport and arrange the polymer-like molecule in the through-hole by a direct method such as optical tweezers.
  • the through hole is relatively long, it is easier to place the polymer molecule in the through hole by sucking the polymer molecule into the through hole. That is, it becomes easier to arrange the polymer-like molecules in the through holes together with the flow of the solution without directly transporting the polymer-like molecules.
  • the minor axis of the cross section perpendicular to the longitudinal direction of the through hole is preferably 1 nm to 1000 nm.
  • the polymer molecule can be kept more stably in the through hole.
  • shear force such as turbulent flow is generated in the solution in the through hole.
  • by making the inside of the through hole a thinner (narrow) space it becomes easier to match the longitudinal direction of the polymer molecule with the longitudinal direction of the through hole.
  • the inside of the through-hole a thinner (narrow) space
  • the kinetic energy due to thermal motion and diffusion of the polymer-like molecule is reduced (entropy of the polymer-like molecule is lowered), and the polymer-like molecule is reduced. Is more stable, and it becomes easier to dispose the polymer-like molecule in the through hole in a state where the polymer molecule is stretched in the longitudinal direction of the through hole.
  • the base material is a substrate having a main surface
  • the shape of the cross section perpendicular to the longitudinal direction of the through hole is substantially elliptical
  • the orientation of the major axis of the elliptical shape it is preferable to be inclined with respect to the main surface.
  • the orientation of the elliptical minor axis and the plane direction of the main surface can be made non-parallel.
  • the base body of the first aspect of the present invention it is preferable that the base body further has a vertical hole communicating the through hole and the outside of the base material. According to this configuration, a liquid containing a gas or a drug is caused to flow into the through hole 1 from the outside of the base material through the vertical hole, and a liquid or gas containing a molecule or a drug in the through hole 1 is supplied. It can be taken out of the substrate.
  • a fixing portion for fixing at least a part of the polymer molecule is provided in the through hole.
  • the fixing part it becomes easier to keep a part of the polymer-like molecule closer to or in contact with the inner wall surface in the through hole.
  • the polymer molecule close to the inner wall surface of the through hole in this way, the kinetic energy due to thermal motion and diffusion of the polymer molecule is further reduced, and the polymer molecule is further stabilized. While maintaining, it becomes much easier to arrange the polymer-like molecules in a state stretched in the longitudinal direction of the through-hole.
  • the fixing portion is made of metal.
  • the portion having a functional group or molecular structure having a high affinity for the metal in the polymer molecule binds to the fixing portion by the fixing portion being a metal, the polymer is fixed to the fixing portion. It becomes easier to fix to.
  • the polymer molecule with the functional group or linking molecule it becomes easier to fix a part of the polymer molecule to the fixing part. As a result, the arrangement of the polymer-like molecules in the through-hole can be controlled more easily.
  • the polymer molecule is preferably DNA, RNA, or polypeptide.
  • the polymer molecule disposed in the through-hole is a polymer composed of a single-stranded or double-stranded DNA molecule, a polymer composed of a single-stranded or double-stranded RNA molecule, or a polymer composed of a polypeptide chain. It is easier to stretch the polymer molecules and arrange them in the through holes in a substantially linear state. For this reason, it becomes easier to analyze the arrangement
  • a base material in which a space for allowing a solution containing polymer-like molecules to flow is provided, and the base material is formed inside the base material, and is open to the space.
  • a through hole having a shape capable of extending and arranging the polymer-like molecule therein, and at least a portion of the base material constituting the through hole is a single member.
  • Step A2 for fixing at least a part of the substrate to the inner wall of the through-hole
  • Step A3 for introducing a binding body binding to the polymer molecule into the through-hole, and a signal generated as a result of the binding to the substrate
  • step A4 to observe from the outside optically, at least.
  • the step A1 at least a part of the polymer molecule introduced into the through hole can be stably maintained in a state of being stretched in the through hole.
  • the subsequent step A2 by fixing at least a part of the polymer molecule to the inner wall of the through hole, the polymer molecule can be prevented from flowing out of the through hole.
  • the combined body is introduced into the through hole.
  • the signal is optically observed.
  • molecules that cause noise include molecules that emit autofluorescence.
  • the polymer-like molecules in the through-holes that are relatively narrow and limited spaces, the number of molecules that become noise sources contained in the spaces is reduced as much as possible, and the observation target is The relative amount of the polymeric molecule and the signal source molecule can be increased. Thereby, a signal with a high S / N ratio can be obtained.
  • the effects described above in the analysis method of the present invention are equivalent to or better than the “optical section effect” obtained using a conventional total reflection microscope or the like, and hereinafter referred to as the “spatial section effect”. .
  • the conjugate is bound to each of a plurality of locations in the polymer molecule, and a plurality of signals resulting from the binding are detected in association with positional information of the plurality of locations. It is preferable to do.
  • the polymer molecule is DNA or RNA
  • the conjugate is a labeled deoxynucleotide
  • the signal is generated by a DNA or RNA replication reaction by a polymerase
  • the optical It is preferable to analyze the base sequence of the DNA or RNA by careful observation.
  • the through-hole can stably arrange a polymer molecule formed of single-stranded or double-stranded DNA or RNA. For this reason, DNA strands or RNA strands longer than 600 to 1000 base pairs used in conventional nucleic acid sequence analysis can be stably arranged and fixed in the through-holes. For this reason, it is possible to stably analyze the base sequence of the DNA chain or RNA chain and obtain more accurate base sequence information.
  • the second solution is preferably passed through the through hole.
  • the polymer molecule is fixed to the inner wall of the through hole. Therefore, even when the second solution is allowed to flow through the through-hole, there is no possibility that the polymer molecule flows out of the through-hole. It is easier to optically observe the signal by controlling the flow of the second solution by including the conjugate or the molecule used for the optical observation in the second solution. It becomes. For example, when the signal is generated by molecules released from the polymer-like molecule at regular intervals, the released molecules are carried on the flow of the second solution, thereby generating signals between the molecules The spatial distance can be increased.
  • the polymer molecule is stretched in the flow direction, and the longitudinal direction of the through-hole and the direction in which the polymer molecule extends are determined. Can be matched. As a result, it becomes easier to optically observe the polymer molecule.
  • the substrate of the present invention it is possible to stably arrange polymer molecules such as DNA strands to be analyzed in the through-holes arranged inside the substrate.
  • the arrangement of the structural units of the polymer-like molecule can be analyzed by using the substrate.
  • FIG. 2 is a cross-sectional view taken along line AA in FIG.
  • FIG. 3 is a schematic diagram showing a state in which polymer molecules T are arranged inside a through hole 1 in the cross-sectional view of FIG. 2. It is a schematic diagram of the cross section orthogonal to the longitudinal direction of the through-hole 1 of FIG. It is a typical perspective view which shows an example of the base
  • FIG. 6 is a cross-sectional view taken along line AA in FIG. 5. It is a typical perspective view which shows an example of the base
  • FIG. 8 is a cross-sectional view taken along line AA in FIG. 7.
  • FIG. 2 is a schematic perspective view showing a laser irradiation method S.
  • FIG. It is a figure which shows typically the relationship between the laser irradiation energy and the modification part (oxygen deficient part) formed. It is a figure which shows typically the relationship between the laser irradiation energy and the modification part (oxygen deficient part) formed.
  • FIG. 1 is a perspective view of a base body 10A that is a first embodiment of a base body according to the present invention.
  • FIG. 2 is a schematic diagram showing a cross section taken along line AA of FIG.
  • FIG. 3 is a schematic diagram showing an example of a state in which the polymer-like molecules are arranged inside the through hole 1 of FIG.
  • the base 10 ⁇ / b> A is provided inside the base 4, the spaces 2 and 3 into which the solution containing polymer molecules flows, and the base 10 ⁇ / b> A formed inside the base 4 and open to the spaces 2 and 3.
  • the through-hole 1 is a substrate on which at least the substrate 4 is arranged, and at least a portion of the substrate 4 constituting the through-hole 4 is formed of a single member, and the through-hole 1 is formed in the inside thereof. It is a shape in which at least a part of the polymer molecule can be stretched and arranged.
  • the spaces 2 and 3 constitute a first flow path 2 and a second flow path 3, respectively.
  • the solution can flow or flow through each channel.
  • the spaces 2 and 3 are not particularly limited as long as the solution can be introduced or circulated.
  • the spaces 2 and 3 constitute flow paths or wells.
  • the shape and volume of the flow channel and well may be appropriately designed according to the amount, viscosity, or various chemical characteristics of the solution to be introduced or circulated.
  • “flowing the solution into the space” means that the solution is introduced (introduced) into the space from the outside of the space.
  • the solution that has flowed in may stay in the space and stay (or be completely stationary), or may flow out of the space. In the latter case, the solution flow is generated in the space by continuously flowing the solution into the space. This applies to all the substrates according to the invention.
  • the through hole 1 is formed inside the substrate 4, one opening of the through hole 1 opens to the side surface 2 a of the first flow path 2, and the other opening of the through hole 1 is the side surface 3 a of the second flow path 3. Open to. That is, the through hole 1 communicates the first flow path 2 and the second flow path 3.
  • the first flow path 2 and the second flow path 3 that are the spaces 2 and 3 are provided on the upper surface 4 a that is the main surface of the base material 4, and face the outside of the base material 4. Therefore, the through hole 1 communicates with the outside of the base material 4 through the first flow path 2 and the second flow path 3.
  • a known fluid control device such as a syringe or a pump is provided in the first flow path 2 and the second flow path 3.
  • a member serving as a lid that covers the first flow path 2 and the second flow path 3 it is possible to send the liquid while applying pressure to the solution using the pump or the like.
  • cover is not drawn in order to make a figure easy to understand.
  • the through-hole is controlled in a controlled direction and a controlled flow rate. 1 can flow in or flow through.
  • a liquid delivery means such as a pump
  • the through-hole is controlled in a controlled direction and a controlled flow rate. 1 can flow in or flow through.
  • the solution is caused to flow into the through-hole 1 from the opening that opens to the first flow path 2, and the second flow path 3 It is possible to make the solution flow out from an opening that opens to the bottom.
  • part which comprises the through-hole 1 is formed with a single member.
  • a single member means that it is different from a member obtained by bonding two or more members by adhesion or the like. That is, the through-hole 1 is formed by perforating a single member, and is not a hole formed by covering a grooved member with a lid.
  • the strength characteristics of the through hole 1 are excellent.
  • the strength characteristics refer to heat resistance, cold resistance, pressure resistance, chemical resistance, and deformation resistance.
  • the substrate 10 of the present invention can withstand heat treatment, cooling treatment, pressurization / negative pressure treatment, chemical treatment, and treatment that causes mechanical deformation. Furthermore, since the site
  • the single member constitutes not only the through hole 1 but also the entire base material 4.
  • the material of the single member include silicon, glass, quartz, and sapphire. Since these materials are excellent in the workability of the through-hole 1, they are preferable. Among these, it is preferable that the material is amorphous so that it is not easily affected by processing anisotropy due to crystal orientation. Furthermore, when observing the inside of the through-hole 1 by optical means such as a microscope, it is more preferable to use glass, quartz, or sapphire because it transmits visible light (wavelength: 0.36 ⁇ m to 0.83 ⁇ m). .
  • a single member constituting the base 4 of the base 10A is a transparent glass substrate.
  • the material of the single member preferably transmits at least part of light having a wavelength of 0.1 ⁇ m to 10 ⁇ m. Specifically, it is preferable to transmit at least part of general light (wavelength of 0.1 ⁇ m to 10 ⁇ m) used as a processing laser. By transmitting such laser light, the modified portion can be formed on the member by laser irradiation as described later. More preferably, the material of the single member transmits light in the visible light region (wavelength of about 0.36 ⁇ m to about 0.83 ⁇ m). By transmitting light in the visible light region, the polymer molecule disposed in the through hole 1 can be observed through the single member using an optical technique such as an optical microscope or a high-resolution CCD camera. .
  • “transmission (transparent)” refers to all states in which light enters the member and transmitted light is obtained from the member.
  • the shape of the through-hole 1 is a shape in which at least a part of the polymer molecule can be stretched and arranged.
  • the stretched polymer molecule is regarded as a linear polymer such as a flexible string (see FIG. 3). That is, the shape of the through hole 1 may be any shape as long as a linear polymer can be disposed therein. For this reason, it is preferable that at least a part of the through hole 1 is columnar.
  • the columnar shape means a three-dimensional shape having a longitudinal direction, and examples thereof include a prism, a polygonal column, a cylinder, and an elliptical column.
  • the longitudinal direction means the height direction with respect to the bottom surface.
  • the polygonal column include a triangular column, a quadrangular column, a pentagonal column, and a hexagonal column.
  • These three-dimensional shapes may include deformations or scratches generated in the formation process of the through-hole 1 other than those strictly defined geometrically. Examples of the deformation include partial or total distortion, extension, or reduction of the three-dimensional shape.
  • the shape of the through hole 1 is preferably an elliptic cylinder, a cylinder, and a quadrangular cylinder.
  • the through holes 1 having these preferable three-dimensional shapes are easier to form.
  • the shape of the inner wall of the through hole 1 having these preferable three-dimensional shapes is relatively simple, it is difficult for turbulent flow to occur in the solution flowing through the through hole 1. For this reason, the polymer molecule can be kept more stable at the columnar portion in the through hole 1.
  • the shape of at least a part of the through-hole 1 is the columnar shape, the direction in which the polymer molecules extend can be arranged along the longitudinal direction of the columnar shape. As a result, it becomes easier to control the arrangement of the polymer molecules in the through-hole 1.
  • the length of the columnar part in the through-hole 1 is not particularly limited, and may be set as appropriate according to the length of the polymer molecule stretched. In that case, it is preferable to make it longer than the stretched length of the polymer molecule from the viewpoint of stably arranging the polymer molecule in the columnar portion.
  • the length of the columnar portion is preferably 100 to 1000%, more preferably 200 to 800% with respect to the length of the polymer molecule.
  • the length of the columnar part is preferably 0.01 ⁇ m to 10 mm, and more preferably 0.1 ⁇ m to 10 mm.
  • the columnar part may be provided at one place of the through hole 1 or may be provided at two or more places. When provided in two or more places, the one columnar portion and the other columnar portion may have the same three-dimensional shape or different three-dimensional shapes.
  • the length in the longitudinal direction (extending direction) of the through-hole 1 is not particularly limited, and may be set as appropriate according to the length of the polymer molecule stretched. At that time, from the viewpoint of stably arranging the polymer molecule in the through-hole 1, it is preferable that the polymer molecule be longer than the stretched length of the polymer molecule.
  • the length in the longitudinal direction is preferably 100 to 1000%, more preferably 200 to 800% with respect to the length of the polymer molecule.
  • the length of the through hole 1 in the longitudinal direction is preferably 0.01 ⁇ m to 10 mm, and more preferably 0.1 ⁇ m to 10 mm.
  • the length of the through hole in the longitudinal direction When the length of the through hole in the longitudinal direction is within the above range, it becomes easier to introduce the polymer molecule into the through hole 1. When it is not less than the lower limit (0.01 ⁇ m) of the range, it becomes easier to suck the polymer molecules together with the solution and arrange them in the through-holes 1. When the length in the longitudinal direction is less than the lower limit, for example, an extremely short length of 1 nm, the polymer molecule flows out of the through hole 1 immediately after the polymer molecule is sucked into the through hole 1. Thus, it may be difficult to place the polymer molecule in the through-hole 1 while being fastened.
  • the upper limit value (10 mm) is not more than the above range, it is possible to prevent an excessive increase in pressure when the solution is sucked or circulated into the through hole, and the polymer molecule is sucked into the through hole 1 together with the solution. It will be easier to do. That is, it becomes easier to arrange the polymer molecules in the through-hole 1 together with the flow of the solution without directly transporting the polymer molecules using optical tweezers or the like.
  • the shape of the cross section orthogonal to the longitudinal direction (extending direction) of the through-hole 1 is not particularly limited, and may be, for example, a shape reflecting the columnar shape, for example, a circle, a substantially circle, an ellipse, a substantially ellipse, a rectangle, or a triangle It can be.
  • the cross-sectional shape is a circle, a substantially circle, an ellipse, a substantially ellipse, a rectangle, or a triangle
  • the diameter, the short diameter, the long diameter, or the length of one side is 0.02 ⁇ m to 5 ⁇ m. be able to.
  • the diameter, the short diameter, or the length of one side can be further reduced.
  • the diameter, the short diameter, or the length of one side can be set to 0.02 to 0.8 ⁇ m, and further can be set to 1 nm to 1000 nm.
  • the short axis (short axis) of the cross section perpendicular to the longitudinal direction of the through hole 1 or the length of the shortest side constituting the circumference of the cross section is between 1 nm and 1000 nm. With this length, the polymer-like molecule can be kept in the through-hole 1 in a more stable and entropically more advantageous state (lower thermal motion or diffusion motion).
  • the shape of the opening where the through hole 1 opens to the first flow path 2 and the second flow path 3 may be any shape, for example, a circle or a substantially circle, an ellipse or a substantially ellipse, a rectangle, or a triangle. can do.
  • the shape of the cross section perpendicular to the longitudinal direction of the through hole 1 may be the same. In this case, the possibility of the turbulent flow of the solution in the through hole 1 can be further reduced, and the polymer molecule can be kept more stable.
  • the through hole 1 is formed to be substantially perpendicular to the side surface 2 a of the first flow path 2 and the side surface 3 a of the second flow path 3.
  • it does not necessarily have to be substantially vertical, and can be freely arranged according to the design of the base 10A.
  • the base material 4 is a substrate having a main surface 4a, the cross-sectional shape orthogonal to the longitudinal direction of the through hole 1 is substantially elliptical, and the orientation of the major axis of the elliptical shape is mainly It is inclined with respect to the surface 4a.
  • the area of the through hole 1 projected onto the main surface 4a is larger, and the inside of the through hole 1 from the main surface 4a is larger. The area that can be observed increases. This point will be described with reference to FIG.
  • FIG. 4 is a schematic diagram showing a cross section of the through hole 1 in the base body 10A perpendicular to the longitudinal direction.
  • the cross-sectional shape is substantially elliptical, and the major axis direction (major axis direction) of the elliptical shape is inclined with respect to the main surface 4 a of the substrate 4.
  • the angle formed by the major axis direction with respect to the main surface 4a is about 30 degrees.
  • the minor axis direction (minor axis direction) of the ellipse is not parallel to the major surface 4a, and the angle formed by the minor axis direction with respect to the major surface 4a is about 60 degrees.
  • the through hole is formed from the main surface 4a side (the direction of the arrow Z). It becomes easier to optically observe the inside of 1. That is, it becomes easier to observe the polymer-like molecule arranged in the through hole 1.
  • a fixing portion for fixing at least a part of the polymer molecule is provided in the through hole 1.
  • the fixing part it becomes easier for a part of the polymer molecules to be kept closer to or in contact with the inner wall surface in the through-hole 1.
  • the kinetic energy due to thermal motion and diffusion of the polymer molecule is further reduced, and the polymer molecule is further stabilized. It is even easier to arrange the polymer-like molecules in a state where they are stretched in the longitudinal direction of the through-hole 1.
  • the fixing part is not particularly limited as long as it has affinity, adsorptivity, or binding property to at least a part of the polymer molecule.
  • fixed part is not restrict
  • the position near the opening of the through hole 1, the end of the through hole 1, or the center of the through hole 1 may be used.
  • the size of the fixing portion and the area of the region where the fixing portion is arranged are not particularly limited, and can be appropriately set according to the diameter of the through hole 1, the type of fixing portion to be used, and the type and length of the polymer molecule. .
  • the size of the fixed portion can be set to, for example, about 0.5 nm to 100 nm.
  • the area of the region where the fixing portion is arranged is provided in, for example, 1 to 100% of the inner wall of the through hole 1.
  • the bond between the fixing part and the polymer molecule may be reversible or irreversible.
  • the fixing portion for example, a pile (metal post) formed of metal or a molecule having specific binding properties is preferable.
  • the metal post examples include nickel, cobalt, magnesium, and gold. It is well known that a thiol group (—SH) can be chemically bonded to the gold. Therefore, for example, one end of the polymer molecule is previously modified with a functional group having a thiol group, and the polymer molecule is disposed in the through hole 1 provided with a metal post formed of gold. In 1, the metal post formed of gold and the functional group are adsorbed to bond and fix the fixing part and one end of the polymer molecule. Further, it is well known that a peptide chain (so-called His-tag) in which six histidines, which are one type of amino acid, are peptide-bonded to nickel and cobalt has a high affinity.
  • His-tag a peptide chain in which six histidines, which are one type of amino acid, are peptide-bonded to nickel and cobalt has a high affinity.
  • one end of the polymer molecule is previously modified with the His tag, and the polymer molecule is disposed in the through hole 1 provided with a metal post formed of nickel or cobalt. Inside, by adsorbing a metal post formed of nickel or cobalt and the histag, the fixing part and one end of the polymer molecule can be bonded and fixed.
  • chelate compounds such as ethylenediaminetetraacetic acid (EDTA) are chemically adsorbed to the magnesium. Therefore, for example, one end of the polymer molecule is previously modified with the EDTA or EDTA derivative, and the polymer molecule is disposed in the through hole 1 having a metal post formed of magnesium. Inside, by adsorbing the metal post formed of magnesium and the EDTA or EDTA derivative, the fixing part and one end of the polymer molecule can be bonded and fixed.
  • a molecule having a specific binding well known among biomolecules, or a linker molecule (linking agent) used in the field of organic chemistry or inorganic chemistry is preferable.
  • Specific examples include avidin, biotin, antibodies against various antigens, and silane coupling agents. It is well known that avidin and biotin bind to each other with high specificity. Therefore, by using one as the fixing part and bonding the other to the polymer molecule in advance, the fixing part and one end of the polymer molecule can be bonded and fixed. The antigen and the antibody can be used in the same manner.
  • the silane coupling agent is well known as a linker molecule that connects an inorganic substance and an organic substance.
  • a method of binding the polymer molecule previously modified with a silane coupling agent to silicon dioxide (silica) constituting glass is well known.
  • a method of chemically introducing a silane coupling agent into a polymer silation method
  • a method of reacting a silane coupling agent with a terminal or a side chain of a polymer, a silane coupling agent together with a monomer constituting the polymer The method of copolymerization is well known.
  • the end of the polymer-like molecule is modified in advance with a known silane coupling molecule, and the polymer-like molecule is provided with a through-hole provided with a fixing portion formed of a material to which the silane coupling molecule can be bonded.
  • the terminal of the polymer molecule can be bonded to the fixing part in the through-hole 1 and fixed.
  • Examples of the polymer molecule disposed in the through-hole 1 of the base body 10A according to the present invention include various known polymer molecules such as a bio-derived polymer and an organic chemically synthesized polymer (resin). It is done. Among these, a polymer derived from a living body is preferable, a polymer composed of a single-stranded or double-stranded DNA molecule, a polymer formed from a single-stranded or double-stranded RNA molecule, or a polymer formed from a polypeptide chain. Is more preferable.
  • these polymer-like molecules can be dissolved in an aqueous solvent, they are easy to handle, and it is easier to arrange the polymer-like molecules in the through-hole 1 in a substantially linear state where the polymer-like molecules are stretched. For this reason, it becomes easier to analyze the arrangement
  • the number, path, and shape of the through holes provided in the base body of the present invention can be appropriately designed according to the purpose of use of the base body or the type and length of the polymer shape disposed inside the through hole.
  • Substrate 10B (10) In the base body 10B according to the present invention shown in FIGS. 5 and 6, three through holes 1 are provided, and two vertical holes 6 are provided in each through hole 1. One end of the vertical hole 6 opens to the through hole 1, and the other end opens to the main surface 4 a of the substrate 4.
  • the solution in the through hole 1 can be circulated through the vertical hole 6. Further, another gas or liquid can be flowed into the through hole 1 or recovered through the vertical hole 6. Therefore, a drug or gas necessary for analysis or the like can be supplied to the polymer molecules arranged in the through hole 1 through the vertical hole 6. As a result, the polymer molecule can be analyzed more easily.
  • the other end of the vertical hole 6 may be opened on a surface other than the main surface 4 a of the substrate 4, for example, a side surface of the substrate 4.
  • the opening can be made at a position required by design. In the case of opening to any surface, the same effect as the case of opening to the main surface 4a is achieved.
  • first through-hole 1 the vertical hole 6 refers to the “second through-hole 6 that communicates the through-hole 1 with the outside of the substrate 4. Can be called.
  • Substrate 10C (10) In the base body 10C according to the present invention shown in FIGS. 7 and 8, the configuration in which the three through holes 1 are provided and the two vertical holes 6 are provided in each through hole 1 is the same as the base body 10B described above. is there. Further, a member (lid material) 5 serving as a lid that covers the main surface 4 a of the base material 4 is provided. On the lower surface of the member 5 facing the main surface 4a, two grooves for connecting a plurality of vertical holes 6 provided in the base material 4 are dug to constitute a third flow path 7 and a fourth flow path, respectively. is doing. With this configuration, the third flow path 7 and the through hole 1 and the fourth flow path and the through hole 1 can be communicated with each other through the vertical holes 6.
  • the third flow path 7 when the third flow path 7 is set to a positive pressure and the fourth flow path 8 is set to a negative pressure, a predetermined liquid or gas flows into the through hole 1 from the third flow path 7 through one vertical hole 6. Furthermore, it can be made to flow from the through hole 1 to the fourth flow path 8 through the other vertical hole 6.
  • the direction in which the liquid or gas in the channel flows is controlled. Therefore, by controlling the third flow path 7, the fourth flow path 8 and the respective vertical holes 6, supply or recovery of chemicals and gases necessary for the analysis etc. with respect to the polymer molecules arranged in the through holes 1 can do. As a result, the polymer molecule can be analyzed more easily. Further, by controlling the third flow path 7, the fourth flow path 8, and each vertical hole 6, the polymer can be effectively allowed to flow into the through hole.
  • the through-hole 1 is provided so as to be bent inside the base material 4 and to have an S shape when viewed from the main surface 4a.
  • the length of the through hole 1 is longer than the through hole of the base body 10A. That is, the through hole 1 can be lengthened without increasing the overall size of the substrate 10. As a result, the longer polymer molecule can be arranged inside the through hole 1.
  • Substrate 10E (10) In the base body 10E according to the present invention shown in FIG. 10, the configuration in which the through-hole 1 is bent inside the base material 4 so as to be S-shaped when viewed from the main surface 4a is the same as the base body 10D described above. It is the same. Further, a plurality of vertical holes 6 are provided in the through hole 1 of the base body 10E. The advantage by having the vertical hole 6 is the same as that of the above-mentioned base
  • Substrate 10F (10) In the base body 10F according to the present invention shown in FIG. 11, the configuration in which the through hole 1 is bent inside the base material 4 so as to have an S shape when viewed from the main surface 4a is the same as that of the base body 10D described above. It is the same. Further, a plurality of landings 9 are provided in the through hole 1 of the base body 10F. By providing the landing 9 in the middle from one end of the through hole 1 to the other end, the flow rate of the solution flowing through the through hole 1 can be relaxed. As a result, the polymer-like molecule disposed in the through hole 1 can be kept more stable. The plurality of landings 9 do not correspond to the columnar part. In the base body 10F, the three cylindrical through holes 1 connected to the two landings 9 correspond to the columnar portion.
  • the through hole 1 is provided to be bent inside the base material 4 so as to be S-shaped when viewed from the main surface 4a.
  • the structure in which the landing 9 is provided is the same as that of the base body 10F described above.
  • a plurality of vertical holes 6 are provided in the through hole 1 of the base body 10G. The advantage by having the vertical hole 6 is the same as that of the above-mentioned base
  • Substrate 10H (10) In the base body 10H according to the present invention shown in FIG. 13, a plurality of temperature control devices 11 are provided in the lower part of the through hole 1 inside the base material 4.
  • Examples of the temperature control device 11 include a heater and a Peltier element. By providing the heater, the temperature in the through hole 1 can be raised. Moreover, the temperature in the through-hole 1 can be lowered
  • the method for disposing the polymer-like molecule inside the through-hole provided in the substrate of the present invention is not particularly limited.
  • a case where the base body 10B (FIG. 5) is used will be described.
  • a solution in which the polymer molecule is contained in a solvent capable of dissolving or dispersing is prepared.
  • the solution can be drawn into the through-hole 1 by causing the solution to flow into the first flow path 2 by a liquid feeding means such as a pump, and then setting the second flow path 3 to a negative pressure.
  • the polymer molecule Since the polymer molecule is contained in the solution drawn into the through hole 1, the polymer molecule is kept in the through hole 1 when the flow of the solution in the through hole 1 is stopped. Can do. At this time, by bringing the polymer molecule into contact with the fixing part, the polymer molecule can be bonded and fixed to the fixing part.
  • the polymer-like molecule may not be fixed after the polymer-like molecule is arranged in the through-hole 1. However, when the solution or a separately prepared solution is circulated in the through-hole 1, the polymer-like molecule may be used. Is preferably fixed in the through-hole 1 to fix the polymer molecule.
  • the polymer-like molecules arranged in the through-hole 1 can be entropically advantageous by being kept in the through-hole 1, that is, a stretched substantially straight string-like state. At this time, by appropriately adjusting the composition of the solution in the through-hole 1, it becomes easier to make the polymer molecule stretched. For example, use of a good solvent or a poor solvent for the polymer-like molecule, or an acidic solvent or an alkaline solvent that can modify the higher-order structure of the polymer-like molecule to form a primary structure.
  • the extraction treatment can be performed using the substrate of the present invention.
  • an opening facing the first flow path 2 of the through hole 1 is obtained by flowing the solution containing the cells U into the first flow path 2 and drawing the solution into the through hole 1 as described above.
  • the cell can be adsorbed to the part.
  • the suction force is further increased, a part of the cell membrane is broken, and the polymer molecules T such as nucleic acids, proteins and sugar chains existing in the cells or on the surface of the cell membrane can be arranged in the through-hole 1. it can.
  • the base 10A to base 10H described above have a configuration in which one or three through holes 1 are provided.
  • the number of through holes, the shape, the path, and the distance between the through holes are not limited to the above example, and can be appropriately designed according to the purpose of use of the substrate.
  • the analysis method of the present invention is a method of analyzing the arrangement of structural units of the polymer-like molecule using the substrate according to the present invention, and includes at least the following steps A1 to A4.
  • the structural unit refers to a molecule corresponding to a monomer constituting the polymer molecule.
  • the polymer molecule is DNA
  • the structural unit is deoxyribonucleotide
  • the sequence of the structural unit is a base sequence of deoxyribonucleotide, that is, adenine (A) guanine (G), cytosine (C ) And thymine (T).
  • the structural unit is a ribonucleotide
  • the sequence of the structural unit is the sequence order of the bases of the ribonucleotide, that is, adenine (A), guanine (G). , Cytosine (C), and uracil (U).
  • the polymer molecule is a protein
  • the structural unit is an amino acid
  • the sequence of the structural unit is a sequence order of 20 or more known amino acids.
  • Step A1 of the analysis method of the present invention is a step of introducing a first solution containing the polymer molecule into the space and introducing the polymer molecule into the through hole.
  • the first solution is drawn into the through hole 1 by flowing the first solution into the first flow path 2, which is the space, and setting the second flow path 3 to a negative pressure. Details of this method are as described above.
  • a method of transporting the polymer molecule into the through-hole 1 using optical tweezers can be exemplified.
  • Step A2 of the analysis method of the present invention is a step of fixing at least a part of the polymer molecule to the inner wall of the through hole.
  • a method of fixing at least a part of the polymer molecule arranged in the through hole 1 in the base body 10B to the inner wall of the through hole for example, using the physicochemical properties inherent in the polymer molecule And a method of adsorbing or bonding to the inner wall of the through hole 1.
  • Examples of the bond that can be generated by the physicochemical property include hydrogen bond, hydrophobic bond (hydrophobic interaction), adsorption by van der Waals force (adsorption by intermolecular force), and the like.
  • a method of fixing via the fixing part is preferable. If the fixing is performed via the fixing portion, the fixing can be performed more reliably. Moreover, the fixed binding force can be adjusted by appropriately selecting the type of the fixing portion. Further, the fixing portion is provided in the through hole 1, and the position where the fixing portion is provided is adjusted.
  • Step A3 of the analysis method of the present invention is a step of introducing a conjugate that binds to the polymer molecule into the through-hole.
  • a conjugate that binds to the polymer molecule fixed to the substrate 10B into the through-hole for example, the conjugate is dissolved or dispersed in a solvent to prepare a solution of the conjugate, and this solution Is allowed to flow into the through hole 1.
  • the combined body can be introduced into the through hole 1.
  • the conjugate binds to the polymer molecule.
  • the conjugate is preferably a labeled deoxynucleotide
  • the signal is preferably generated by a DNA or RNA replication reaction by DNA polymerase or RNA polymerase.
  • the analysis target is DNA
  • the analysis target is DNA
  • the analysis target is DNA
  • the analysis target is RNA
  • the analysis target when the analysis target is RNA, it can be analyzed in the same manner as when the analysis target is DNA by using RNA-dependent RNA polymerase (RNA replicase) and labeled ribonucleotide instead of DNA polymerase and labeled deoxyribonucleotide.
  • RNA-dependent RNA polymerase RNA replicase
  • labeled ribonucleotide instead of DNA polymerase and labeled deoxyribonucleotide.
  • Step A4 of the analysis method of the present invention is a step of optically observing a signal generated as a result of the binding from the outside of the substrate.
  • a primer having a known sequence or a random primer is bound to a predetermined position of the DNA strand, and the polymerase is bound to the DNA strand.
  • a plurality of polymerases may be bound to one DNA strand.
  • labeled dATP deoxyadenosine triphosphate
  • labeled dGTP deoxyguanosine triphosphate
  • labeled dCTP deoxycytidine triphosphate
  • labeled dTTP deoxythymidine triphosphate
  • dNTP complementary labeled deoxynucleotide
  • the signal is obtained from the location where the polymerase is bound, when the primer, polymerase, and dNTP are bound to a plurality of locations on a single DNA, a plurality of signals generated from the respective locations are converted into position information and In association, detection can be performed during unit time.
  • detection can be performed during unit time.
  • four types of fluorescent signals can be observed. Therefore, as the DNA replication reaction proceeds, fluorescence signals corresponding to the base sequence of the DNA strand are observed sequentially.
  • the base sequence of the DNA strand can be obtained by measuring and analyzing the type, generation order, and position of the fluorescent signal.
  • the FRET system disclosed in Patent Document 1 can be applied to the method for generating a fluorescent signal associated with the DNA replication reaction without departing from the gist of the present invention. Further, the position of the signal can be determined with sub-nanometer accuracy by using a known position determination method such as FIONA (Fluorescence Imaging One-Nanometer Accuracy). Thereby, the difference in the position of one base can be detected.
  • FIONA Fluorescence Imaging One-Nanometer Accuracy
  • the method of optically observing the fluorescence signal generated as a result of binding of the labeled deoxynucleotide as the conjugate to the DNA strand as the polymer molecule in the DNA replication reaction by the polymerase from the outside of the substrate 10B is particularly limited.
  • a high-resolution CCD camera used in combination with an optical microscope can be used.
  • the polymer molecule When the polymer molecule is a protein, it is denatured into a linear polypeptide and introduced into the through-hole 1 in the presence of a known denaturant. Thereafter, a buffer solution or the like under physiological conditions is introduced to remove the denaturing agent. Next, an antibody, DNA / RNA aptamer, or organic compound that recognizes 3 to 4 amino acids is introduced. These substances are called amino acid determining molecules. These strictly identify (recognize) only the first amino acid, and the remaining 2 to 3 residues may be bound to any kind of amino acid. Only such types of amino acid determining molecules corresponding to all amino acids are prepared and sequentially introduced into the through hole 1.
  • These molecules are fluorescently labeled, and a signal derived from an amino acid determinant molecule bound on the stretched polypeptide can be detected by using a fluorescence microscope or a fluorescence detector.
  • the obtained signal includes a plurality of the same amino acids in the polypeptide. Therefore, a plurality of detections are simultaneously made at predetermined positions corresponding to the amino acids constituting the polypeptide.
  • the position of the fluorescence signal is determined with sub-nanometer accuracy using a known position determination method such as FIONA. Thereafter, heat or a denaturant is introduced to remove the amino acid determinant molecules bound thereto.
  • Examples of the method for producing the “antibody recognizing 3 to 4 amino acids” include the following methods.
  • the first one is uniquely determined as the type (degree of freedom) of the amino acid sequence formed by the 4 residues recognized by the antibody.
  • 20 to the third power 8000 types.
  • 8000 kinds of peptides can be prepared by determining only the first one amino acid as one kind and then chemically synthesizing the subsequent sequences randomly.
  • the limit of the library size in chemical synthesis is said to be about 10 to the 8th power (10 8 ), so it is theoretically possible to cover all of peptides composed of 6 random amino acids. .
  • the C-terminus of the synthesized peptide is immobilized on latex beads via an appropriate linker such as PEG (polyethylene glycol).
  • an animal such as a mouse or a rabbit is immunized by a conventional method to obtain a polyclonal antibody.
  • the type of antibody obtained depends on the number of B cells possessed by the animal to be immunized. Normally, B cells are present in the order of about 10 9 (10 9 ) to 10 10 (10 10 ), so that antibodies capable of recognizing all 8000 types can be produced with sufficient redundancy.
  • an antibody capable of binding to an amino acid sequence formed by 4 residues having the first determined “one kind” amino acid at the beginning can be obtained.
  • a polyclonal antibody can be obtained by using 8000 types of antigens for each of the remaining 19 types of amino acids.
  • a total of 20 lots of polyclonal antibodies may be prepared corresponding to 20 kinds of amino acids.
  • the amino acid sequence of the polypeptide can be determined.
  • Step M1 for forming the mass portion 51
  • Step M2 for forming the first flow path 2 and the second flow path 3 forming the space in the single member 4
  • a single member 4 and at least a step M3 for removing the modified portion 51 by etching.
  • the laser L laser light L
  • a laser light having a pulse width of a pulse time width of picosecond order or less For example, a titanium sapphire laser, a fiber laser having the pulse width, or the like can be used.
  • the laser L laser light L
  • light in a general wavelength region 0.1 to 10 ⁇ m
  • the member 4 which is a workpiece.
  • the modified portion 51 can be formed in the member 59.
  • Examples of the material of the member 4 include silicon, glass, quartz, and sapphire. These materials are preferable because they are excellent in workability when forming the through hole 1.
  • the material is preferably amorphous so as not to be affected by processing anisotropy due to crystal orientation.
  • the material of the member 4 transmits light having at least some wavelengths among light having wavelengths of 0.1 ⁇ m to 10 ⁇ m. Specifically, it is preferable to transmit at least a part of light in a general wavelength region (0.1 ⁇ m to 10 ⁇ m) used as a processing laser beam.
  • the modified portion can be formed by irradiating the member with laser as described later.
  • the material of the member 4 is transparent to light in the visible light region (wavelength of about 0.36 ⁇ m to about 0.83 ⁇ m).
  • the material of the member 4 transmits light in the visible light region, the polymer molecule introduced into the through hole 1 can be optically observed.
  • the single member 4 is a transparent glass substrate (hereinafter referred to as a glass substrate 4).
  • a glass substrate 4 a transparent glass substrate
  • Silicon, quartz, and glass are more suitable for the workability in the process M2 to be described later.
  • the glass substrate 4 for example, a glass substrate formed of quartz, a glass substrate mainly composed of silicate, a glass substrate formed of borosilicate glass, or the like can be used.
  • a glass substrate formed of synthetic quartz is preferable because of good workability.
  • the thickness of the glass substrate 4 is not particularly limited.
  • the modified part 51 in which the glass is modified is formed by irradiating the laser beam L so as to be focused and focused on the inside of the glass substrate 4 and scanning the focal point in the direction of the arrow.
  • the modified portion 51 having a desired shape can be formed.
  • the “modified portion” means a portion having low etching resistance and selectively or preferentially removed by etching.
  • the irradiation intensity is set to a value close to the processing upper limit threshold (processing appropriate value) of the glass substrate 4 or less than the processing upper limit threshold, and the polarization direction (electric field direction) of the laser light L is set to the scanning direction. It is preferable to be perpendicular to the surface.
  • this laser irradiation method is referred to as a laser irradiation method S.
  • the laser irradiation method S will be described with reference to FIG.
  • the propagation direction of the laser light L is an arrow Z
  • the polarization direction (electric field direction) of the laser light L is an arrow Y.
  • the irradiation region of the laser light L is set within a plane 50 formed by the propagation direction Z of the laser light and a direction perpendicular to the polarization direction of the laser light.
  • the laser irradiation intensity is set to a value close to the processing upper limit threshold of the glass substrate 4 or less than the processing upper limit threshold.
  • the modified portion 51 having a nano-order aperture can be formed in the glass substrate 4.
  • the modified portion 51 having a substantially elliptical cross section with a minor axis of about 20 nm and a major axis of about 0.2 ⁇ m to 5 ⁇ m is obtained.
  • the direction along the laser propagation direction is the major axis
  • the direction along the laser electric field direction is the minor axis.
  • the cross section may have a shape close to a rectangle.
  • the obtained modified portion 51 may be formed with a periodic structure.
  • a periodic structure having periodicity along the direction may be formed in a self-forming manner.
  • the formed periodic structure is a layer with low etching resistance.
  • the oxygen-deficient layer and the oxygen-enriched layer are periodically arranged (FIG. 17B), and the etching resistance of the oxygen-deficient portion is weakened. A portion can be formed. Such periodic recesses and protrusions are not necessary in the formation of the through holes 1 described later.
  • the laser irradiation intensity is less than the processing upper limit threshold value of the glass substrate 4 and the laser irradiation intensity can be reduced by reducing the etching resistance by modifying the glass substrate 4.
  • the periodic structure is not formed, and one oxygen-deficient portion (a layer having low etching resistance) is formed by laser irradiation (FIG. 17A). Further, when the one oxygen-deficient portion is etched, one through hole 1 can be formed.
  • the shape of the cross section perpendicular to the longitudinal direction of the through-hole 1 can be an ellipse or a substantially ellipse.
  • the minor axis can be controlled to a nano-order size by etching.
  • microorganisms can be captured by making the minor axis smaller than, for example, the microorganism size. At this time, since the major axis can be made larger than the nano-order size, the pressure loss of the fluid flowing into the through hole 1 can be reduced.
  • the through-hole 1 it is preferable to fill the through-hole 1 with a solution in advance as a preparation for introducing the polymer molecule or a preparation for capturing cells and microorganisms.
  • a solution in advance as a preparation for introducing the polymer molecule or a preparation for capturing cells and microorganisms.
  • the capillary force increases as the through hole becomes finer, there may be a problem that the solution does not come out of the through hole 1 from the through hole 1.
  • the major axis is made sufficiently large even if the polymer molecule is introduced or the minor axis is sufficient to capture the microorganism.
  • the capillary force can be suppressed, and the adverse effect that the solution does not come out of the through hole 1 can be suppressed.
  • the oxygen-deficient portion 51 in this specification Even when a layer with low etching resistance (oxygen-deficient portion in quartz or glass) is formed by a single layer by laser irradiation (referred to as the modified portion 51 in this specification), the oxygen-deficient portion is extremely selective for etching. It becomes a layer with high properties. This has been found by the inventors' diligent study.
  • the processing upper limit threshold is defined as the lower limit value of the laser pulse power at which the periodic structure can be formed (upper limit value in the range of laser pulse power at which the periodic structure is not formed).
  • the “lower limit (threshold value) of laser irradiation intensity that can reduce the etching resistance by modifying the glass substrate 4” is a limit value at which the through-hole 1 can be formed in the glass substrate 4 by the etching process. is there. If it is lower than this lower limit value, a layer having low etching resistance cannot be formed by laser irradiation, and therefore there is no through hole 1.
  • the “processing upper limit threshold” means an electron plasma wave generated by the interaction between the base material and the laser light at the focal point (condensing region) of the laser light irradiated into the base material. It means the lower limit value of the laser irradiation intensity at which interference with the incident laser beam occurs, and due to the interference, a striped modified portion can be formed in a self-forming manner.
  • the “processing lower limit threshold (threshold value)” means a modified portion obtained by modifying the base material at the focal point (condensing area) of the laser light irradiated into the base material.
  • the lower limit value of the laser irradiation intensity that can reduce the etching resistance of the modified portion to such an extent that it can be formed and selectively or preferentially etched by the subsequent etching process.
  • a region irradiated with laser with a laser irradiation intensity lower than the lower limit value is difficult to be selectively or preferentially etched in the subsequent etching process. For this reason, in order to form a modified portion that becomes a fine hole after etching, it is preferable to set the laser irradiation intensity to be equal to or higher than the processing lower limit threshold.
  • the processing upper limit threshold (process appropriate value) and the processing lower limit threshold (threshold) are generally determined by the wavelength of the laser light, the material (material) of the substrate that is the target of laser irradiation, and the laser irradiation conditions. However, when the relative directions of the polarization direction of the laser beam and the scanning direction are different, the processing upper limit threshold and the processing lower limit threshold may be slightly different. For example, the processing upper limit threshold and the processing lower limit threshold may differ between when the scanning direction is perpendicular to the polarization direction and when the scanning direction is parallel to the polarization direction. Therefore, the processing upper limit threshold and the processing lower limit threshold when the relative relationship between the polarization direction of the laser light and the scanning direction is changed in the wavelength of the laser light to be used and the base material to be used are examined in advance. It is preferable.
  • the method of scanning the focal point of the laser beam L is not particularly limited, but the modified portion 51 that can be formed by one continuous scanning is a direction perpendicular to the propagation direction Z of the laser beam L and the polarization direction Y of the laser beam L. It is limited within the plane 50 comprised by these. If it exists in this plane 50, the shape of the modification part formed can be adjusted.
  • the propagation direction of the laser light L is shown as being perpendicular to the upper surface of the glass substrate 4, but is not necessarily perpendicular.
  • the laser L may be irradiated at a desired incident angle with respect to the upper surface.
  • the cross-sectional shape orthogonal to the propagation direction of the laser light L and the longitudinal direction of the modified portion 51 is the substantially ellipse, the major axis direction of the ellipse and the propagation direction of the laser light L substantially coincide. Therefore, as shown in FIG.
  • the propagation direction of the laser beam L that is, the irradiation angle is set to the upper surface (main Irradiation may be performed with a desired angle with respect to the surface 4a).
  • the laser transmittance of the modified part is different from the laser transmittance of the unmodified part. For this reason, it is usually difficult to control the focal position of the laser light transmitted through the modified portion. Therefore, it is desirable to form the modified portion from a region located in the back as viewed from the surface on the laser irradiation side.
  • the modified portion 51 may be formed by condensing the laser light L with a lens and irradiating it as described above.
  • a lens for example, a refractive objective lens or a refractive lens can be used, but it is also possible to irradiate with, for example, Fresnel, reflection type, oil immersion, water immersion type.
  • a cylindrical lens it is possible to irradiate a wide area of the glass substrate 4 with a laser at a time.
  • the laser beam L can be irradiated at once in a wide range in the vertical direction of the glass substrate 4.
  • the polarization of the laser light L needs to be horizontal with respect to the direction in which the lens has a curvature.
  • the laser irradiation condition S include the following various conditions.
  • a titanium sapphire laser (a laser using a crystal in which sapphire is doped with titanium as a laser medium) is used.
  • the laser light to be irradiated for example, a wavelength of 800 nm and a repetition frequency of 200 kHz are used, and the laser light L is condensed and irradiated at a laser scanning speed of 1 mm / second.
  • These values of wavelength, repetition frequency, and scanning speed are examples, and the present invention is not limited to this, and can be changed as necessary.
  • the pulse intensity when irradiating the glass substrate is preferably a value close to the processing upper limit threshold, for example, a power of about 80 nJ / pulse or less. If the power is higher than that, a periodic structure is formed and they are connected by etching, so that it is difficult to form the through-hole 1 having a nano-order diameter. In some cases, the diameter becomes a micron order, or the periodic structure is formed. N. Processing is possible even if A. ⁇ 0.7, but the spot size becomes smaller and the laser fluence increases, so that laser irradiation with a smaller pulse intensity is required.
  • Step M2 Next, the first flow path 2 and the third flow path 3 that form the space are formed on a single glass substrate 4.
  • a resist 52 is patterned and arranged on the upper surface of the glass substrate 4 by, for example, photolithography (FIG. 15B).
  • the region where the resist 52 is not provided on the upper surface of the glass substrate 4 is etched and removed until reaching a predetermined depth by a method such as dry etching, wet etching, or sand blasting (FIG. 15C).
  • a method such as dry etching, wet etching, or sand blasting
  • step M2 it is preferable to expose the cross section of the modified portion 51 formed in step M1 on the side surface 2a of the first flow path 2 and the side surface 3a of the second flow path 3 to be formed. By doing so, it becomes easier to form the through hole 1 by the etching process in the subsequent step M3.
  • Step M3 the modified portion 51 formed in step M1 is removed from the single glass substrate 4 by etching (FIG. 15D).
  • etching As an etching method, wet etching is preferable.
  • the modified portion 51 having a cross section exposed to the side surface 2a of the first flow path 2 and the side surface 3a of the second flow path 3 has low etching resistance, and can be selectively or preferentially etched.
  • This etching utilizes the phenomenon that the modified portion 51 is etched much faster than the unmodified portion of the glass substrate 4. As a result, the through hole 1 corresponding to the shape of the modified portion 51 is used. Can be formed.
  • the etching solution is not particularly limited, and for example, a solution containing hydrofluoric acid (HF) as a main component, or a hydrofluoric acid-based mixed acid obtained by adding an appropriate amount of nitric acid or the like to hydrofluoric acid can be used. Also, other chemicals can be used depending on the material of the member 4.
  • the through-hole 1 having a nano-order diameter can be formed at a predetermined position in the glass substrate 4 so as to communicate the first flow path 2 and the second flow path 3.
  • the size of the through-hole 1 can be, for example, a through-hole having a substantially elliptical cross section with a minor axis of about 20 nm to 200 nm and a major axis of about 0.2 ⁇ m to 5 ⁇ m.
  • the cross section may be a shape close to a rectangle.
  • the size difference between the modified portion 51 and the through hole 1 can be reduced or increased. It is theoretically possible to make the minor axis several nanometers to several tens of nanometers by shortening the treatment time. On the contrary, by increasing the processing time, the minor axis can be set to about 1 ⁇ m to 2 ⁇ m and the major axis can be set to about 5 ⁇ m to 10 ⁇ m.
  • a member serving as a lid may be bonded to the upper surface of the glass substrate 4 so as to cover the upper surfaces of the formed first flow path 2 and second flow path 3 as necessary.
  • the method for bonding the member to be the lid and the upper surface of the glass substrate 4 may be performed by a known method according to the material of the member to be the lid.
  • the material of the member to be the lid is not particularly limited, and a resin substrate such as PDMS or PMMA, or a glass substrate can be used. Moreover, it is preferable that the material of the member used as the lid transmits light (for example, visible light) of the optical observation means.
  • wet etching or dry etching can be applied.
  • wet etching it is most preferable to use, for example, 1% or less hydrofluoric acid, but other acid or basic containers may be used.
  • isotropic etching methods include various dry etching methods such as barrel type plasma etching, parallel plate type plasma etching, and downflow type chemical dry etching.
  • anisotropic dry etching method for example, a parallel plate type RIE, a magnetron type RIE, an ICP type RIE, an NLD type RIE, etc. can be used as reactive ion etching (hereinafter referred to as RIE).
  • RIE reactive ion etching
  • a process close to isotropic etching can be performed by shortening the mean free path of ions by a method such as increasing the process pressure.
  • the gas used is mainly a gas capable of chemically etching materials such as fluorocarbon, SF, CHF3, fluorine gas, and chlorine gas, and other gases such as oxygen, argon, and helium are appropriately used. They can be mixed and used, and can be processed by other dry etching methods.
  • step M2 a more preferable etching is anisotropic etching, and in step M3, a more preferable etching is isotropic etching.

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  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Plasma & Fusion (AREA)
  • Mechanical Engineering (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Selon la présente invention, au moins un matériau de base, des espaces, et des trous de passage sont disposés dans le substrat. Les espaces sont disposés dans le matériau de base et une solution contenant une molécule polymère est versée dans ceux-ci. Les trous de passage sont formés à l'intérieur du matériau de base, débouchent sur les espaces et ont une forme telle que les molécules polymères puissent être allongées et disposées dans ceux-ci. Dans le matériau de base, au moins la zone configurant les trous de passage est réalisée sous la forme d'un composant unique.
PCT/JP2012/072051 2011-08-30 2012-08-30 Substrat et procédé d'analyse WO2013031912A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013220958A (ja) * 2012-04-13 2013-10-28 Namiki Precision Jewel Co Ltd 微小空洞形成方法

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JP2007159439A (ja) * 2005-12-12 2007-06-28 Quantum 14:Kk ポーラスシリコンにより高密度反応空間アレイを形成したマイクロリアクター
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JP2008288577A (ja) * 2007-04-18 2008-11-27 Fujikura Ltd 基板の処理方法、貫通配線基板及びその製造方法、並びに電子部品
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JP2007159439A (ja) * 2005-12-12 2007-06-28 Quantum 14:Kk ポーラスシリコンにより高密度反応空間アレイを形成したマイクロリアクター
JP2007304055A (ja) * 2006-05-15 2007-11-22 Dna Chip Research Inc ポーラスシリコン基板上にポリヌクレオチドが固定化されてなるマイクロアレイ
JP2008288577A (ja) * 2007-04-18 2008-11-27 Fujikura Ltd 基板の処理方法、貫通配線基板及びその製造方法、並びに電子部品
WO2012008577A1 (fr) * 2010-07-16 2012-01-19 株式会社フジクラ Substrat et son procédé de production

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013220958A (ja) * 2012-04-13 2013-10-28 Namiki Precision Jewel Co Ltd 微小空洞形成方法

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