WO2013031912A1

WO2013031912A1 - Substrate and analysis method

Info

Publication number: WO2013031912A1
Application number: PCT/JP2012/072051
Authority: WO
Inventors: 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
Priority date: 2011-08-30
Filing date: 2012-08-30
Publication date: 2013-03-07
Also published as: JPWO2013031912A1; JP6134645B2

Abstract

At least a base material, spaces, and through holes are disposed in the substrate. The spaces are provided in the base material and a solution containing a polymeric molecule is poured therein. The through holes are formed inside the base material, are open to the spaces and have a shape such that the polymeric molecules can be elongated and disposed therein. In the base material, at least the area configuring the through holes is formed as a single member.

Description

Substrate and analysis method

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.
This application claims priority based on Japanese Patent Application No. 2011-187510 filed in Japan on August 30, 2011, the contents of which are incorporated herein by reference.

A complete version of the entire DNA base sequence constituting the human genome was released in 2003 by the Human Genome Project aimed at decoding all human genetic information. The total length of this DNA base sequence is as long as 3 billion base pairs, and it is said that the length is about 2 meters when stretched straight. The above-described human genetic information has been deciphered with the help of an international human genome sequence consortium, spending more than 10 years and a huge amount of money. Even after the completion of the Human Genome Project, the development of DNA base sequence analysis methods and analyzers (DNA sequencers) are steadily progressing. The goal is to develop a system that can analyze an individual's human genome in less than $ 1000 in a few days. This system is thought to be able to provide tailor-made medical care tailored to individual genetic characteristics.

In recent years, the analysis speed of DNA sequencers has been dramatically improved. If the DNA sequencer used in the human genome project is the first generation, next-generation and next-generation analyzers have been developed and proposed one after another. In addition to the speed, a technique for improving the accuracy of sequence information is being developed. For example, 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.

JP 2010-142250 A

In the method disclosed in Patent Document 1, DNA synthase is immobilized on the surface of a glass substrate, a DNA strand replication reaction is caused in the vicinity of the substrate surface, and the fluorescence of the product is optically observed. By doing so, the base sequence of the DNA strand is read. At this time, in order to increase the signal / noise ratio (S / N ratio) of fluorescence measurement, a complicated and expensive apparatus (total reflection illumination fluorescence microscope) capable of generating excitation light only near the substrate surface is necessary. However, even when this apparatus is used, since evanescent light is used as excitation light, observation conditions are severely limited, and there is a possibility that signals cannot be sufficiently detected. In general, long DNA strands are easily cleaved by a physical shearing force. At the time of analysis, 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.

According to the substrate of the first aspect of the present invention, 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 | part which comprises the said through-hole is formed with a single member, and there is no location and seam bonded together in the said through-hole, it is excellent in the intensity | strength of the said through-hole. Here, the strength refers to heat resistance, cold resistance, pressure resistance, chemical resistance, and deformation resistance. Furthermore, since the site | 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 | occur | produced in the light for observation irradiated into the through-hole, or in a through-hole There is no possibility that the optical signal is refracted and becomes stray light. For this reason, it is easy to optically observe the inside of the through hole.

In the substrate according to the first aspect of the present invention, it is preferable that at least a part of the through hole is columnar.
When 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.

In the substrate according to the first aspect of the present invention, the length of the through hole in the longitudinal direction is preferably 0.1 μm to 10 mm.
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. When 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. On the other hand, when 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.

In the substrate according to the first aspect of the present invention, the minor axis of the cross section perpendicular to the longitudinal direction of the through hole is preferably 1 nm to 1000 nm.
When the minor axis of the cross section perpendicular to the longitudinal direction of the through hole is within the above range, the polymer molecule can be kept more stably in the through hole. By being in the range, it is possible to further reduce the possibility that shear force such as turbulent flow is generated in the solution in the through hole. In addition, 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. Furthermore, by making 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.

In the substrate of the first aspect of the present invention, 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, and the orientation of the major axis of the elliptical shape However, it is preferable to be inclined with respect to the main surface.
According to this configuration, the orientation of the elliptical minor axis and the plane direction of the main surface can be made non-parallel. By providing the through hole in the base so as to have such a relative relationship, it becomes easier to optically observe the inside of the through hole from the main surface side. As can be understood from the fact that the projected area of the through hole on the main surface becomes larger, the area where the inside of the through hole can be observed is expanded from the main surface.

In 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.

In the substrate of the first aspect of the present invention, it is preferable that a fixing portion for fixing at least a part of the polymer molecule is provided in the through hole.
By providing 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. By keeping 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.

In the base according to the first aspect of the present invention, it is preferable that the fixing portion is made of metal.
By utilizing the fact that 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. By partially modifying 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.

In the substrate of the first aspect of the present invention, the polymer molecule is preferably DNA, RNA, or polypeptide.
When 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 | sequence of the structural unit (monomer) which comprises the said polymer-like molecule | numerator.

In the analysis method of the second aspect of the present invention, 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. Using the substrate of the first embodiment to be formed, allowing the first solution containing the polymer molecule to flow into the space and introducing the polymer molecule into the through-hole; and the polymer molecule 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 Including the step A4 to observe from the outside optically, at least.

According to the analysis method of the present invention, in 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. In 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. Thereafter, in the step A3, the combined body is introduced into the through hole. In the next step A4, the signal is optically observed. At this time, since the inside of the through-hole contains only a limited amount of solution, there is only a limited amount of molecules serving as noise sources contained in the solution. Examples of molecules that cause noise include molecules that emit autofluorescence. Thus, by arranging 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”. .

In the analysis method of the second aspect of the present invention, 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.

In the analysis method of the second aspect of the present invention, 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, and 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.

In the analysis method of the second aspect of the present invention, the second solution is preferably passed through the through hole.
In the step A2, 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. As a result, it becomes easier to distinguish a plurality of signals and detect each of them. In addition, by causing the second solution to flow in one direction of the through-hole, 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.

According to 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. According to the analysis method of the present invention, the arrangement of the structural units of the polymer-like molecule can be analyzed by using the substrate.

It is a typical perspective view which shows an example of the base | substrate concerning this invention. 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 | substrate concerning this invention. 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 | substrate concerning this invention. FIG. 8 is a cross-sectional view taken along line AA in FIG. 7. It is a typical perspective view which shows an example of the base | substrate concerning this invention. It is a typical perspective view which shows an example of the base | substrate concerning this invention. It is a typical perspective view which shows an example of the base | substrate concerning this invention. It is a typical perspective view which shows an example of the base | substrate concerning this invention. It is a typical perspective view which shows an example of the base | substrate concerning this invention. It is a typical perspective view which shows an example of the base | substrate concerning this invention. It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate concerning this invention. It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate concerning this invention. It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate concerning this invention. It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate concerning this invention. 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.

The present invention will be described below based on preferred embodiments with reference to the drawings.
<Substrate>
[Substrate 10A (10)]
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.

In the upper surface (main surface) 4 a of the base material 4, 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. For example, 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.

Here, “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.

As a method for allowing the liquid to flow into or flow through the first flow path 2 and the second flow path 3, a known fluid control device (not shown) such as a syringe or a pump is provided in the first flow path 2 and the second flow path 3. Just connect. By disposing 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. In addition, in FIG. 1, the member used as the said lid | cover is not drawn in order to make a figure easy to understand.
In this way, by controlling the flow of the solution in the first flow path 2 and the second flow path 3 by using 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. For example, by setting the first flow path 2 to a positive pressure and the second flow path 3 to a negative pressure, 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.

The site | part which comprises the through-hole 1 is formed with a single member. Here, 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. In the through hole 1 of the present invention, since there is no place or joint where two or more members are bonded together, the strength characteristics of the through hole 1 are excellent. Here, the strength characteristics refer to heat resistance, cold resistance, pressure resistance, chemical resistance, and deformation resistance. Since the strength characteristics are thus excellent, 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 | part which comprises the through-hole 1 is formed with a single member, and there is no location or joint in the through-hole 1, the light for analysis irradiated into the through-hole 1 and the inside of the through-hole 1 The optical signal generated in step 1 is not refracted by the bonded portion or seam. For this reason, it is easy to optically observe and analyze the inside of the through hole 1.

In the base body 10 </ b> A shown in FIG. 1, the single member constitutes not only the through hole 1 but also the entire base material 4.
Examples of 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 μm to 0.83 μm). . 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 μm to 10 μm.
Specifically, it is preferable to transmit at least part of general light (wavelength of 0.1 μm to 10 μm) 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 μm to about 0.83 μm). 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. .
In the present invention, “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. Usually, 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. In these columnar shapes, the longitudinal direction means the height direction with respect to the bottom surface. Examples of 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.
In 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. Moreover, since 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.
When 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. For example, 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. Specifically, the length of the columnar part is preferably 0.01 μm to 10 mm, and more preferably 0.1 μm 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. For example, 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. Specifically, the length of the through hole 1 in the longitudinal direction is preferably 0.01 μm to 10 mm, and more preferably 0.1 μm to 10 mm.

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 μm) 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.
When 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. Specifically, when 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 μm to 5 μm. be able to. By forming the through hole 1 by a method described later, the diameter, the short diameter, or the length of one side can be further reduced. For example, the diameter, the short diameter, or the length of one side can be set to 0.02 to 0.8 μm, and further can be set to 1 nm to 1000 nm.

It is preferable that 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. Specifically, for example, 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.

2 and 3, 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. However, it does not necessarily have to be substantially vertical, and can be freely arranged according to the design of the base 10A.
Moreover, it is also possible to process in the shape of a funnel by slightly widening the diameter of the opening of the through hole 1 slightly than the diameter of the through hole 1. By processing in this way, when the polymer molecule is introduced into the through-hole 1, it is possible to reliably prevent the polymer molecule from being unnecessarily caught on the edge of the opening.

In the base body 10A according to the present invention, 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. As a result, compared with the case where the direction of the major axis is perpendicular to the main 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. In the example shown in FIG. 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.
Thus, by adjusting the relative positional relationship between the major axis and the main surface 4a and providing the through hole 1 in the base material 4, 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.

In the base body 10A according to the present invention, it is preferable that a fixing portion for fixing at least a part of the polymer molecule is provided in the through hole 1.
By providing 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. Thus, by keeping the polymer molecule close to the inner wall surface of 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. In the through-hole 1, the position in particular which provides the said fixing | fixed part is not restrict | limited, It can set suitably according to the position which fixes the said polymer molecule.
For example, 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. . Specifically, the size of the fixed portion can be set to, for example, about 0.5 nm to 100 nm. In addition, 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.

As the fixing portion, for example, a pile (metal post) formed of metal or a molecule having specific binding properties is preferable.

Examples of the metal post 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. Therefore, for example, 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.
It is well known that 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.

As the molecule having the specific binding property, for example, 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. is there. 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. For example, a method of binding the polymer molecule previously modified with a silane coupling agent to silicon dioxide (silica) constituting glass is well known. As a method of chemically introducing a silane coupling agent into a polymer (silylation 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. Therefore, for example, 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.
When fixing the inner wall of the through-hole 1 and the polymer-like molecule using a silane coupling agent as a linker molecule, the silane coupling molecule previously introduced into the polymer-like molecule with respect to the glass constituting the inner wall of the through-hole 1 May be directly coupled. In this fixation, the silane coupling molecule corresponds to the fixing part.

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. Since 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 | sequence of the structural unit (monomer) which comprises the said polymer-like molecule | numerator.

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. By providing the vertical hole 6, 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. In addition, by individually controlling the pressure in each vertical hole 6, it is possible to effectively cause the polymer to flow into the through hole.

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. If the through-hole 1 for introducing the polymer molecule is referred to as “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.
Accordingly, 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. By adjusting the pressure in each channel, 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.

[Substrate 10D (10)]
In the base body 10D according to the present invention shown in FIG. 9, 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. As is apparent from the figure, 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 10B.

[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.

[Substrate 10G (10)]
In the base body 10G according to the present invention shown in FIG. 12, 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. Further, 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 10B.

[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 | hung by providing a Peltier element. Thus, since the temperature in the through-hole 1 can be controlled by the temperature control device 11, it becomes easier to analyze the polymer molecule.

<Method of arranging polymer-like molecules in the through-hole>
The method for disposing the polymer-like molecule inside the through-hole provided in the substrate of the present invention is not particularly limited. For example, a case where the base body 10B (FIG. 5) is used will be described.
First, a solution in which the polymer molecule is contained in a solvent capable of dissolving or dispersing is prepared.
Next, 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. 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.

Further, when the polymer molecule is extracted from a microorganism or a cell, the extraction treatment can be performed using the substrate of the present invention. As shown in FIG. 14, 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. At this time, when 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. In the substrate according to the present invention, 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.

<Method for analyzing polymer-like molecules>
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.
Here, the structural unit refers to a molecule corresponding to a monomer constituting the polymer molecule. For example, when the polymer molecule is DNA, the structural unit is deoxyribonucleotide, and the sequence of the structural unit is a base sequence of deoxyribonucleotide, that is, adenine (A) guanine (G), cytosine (C ) And thymine (T). For example, when the polymer molecule is RNA, the structural unit is a ribonucleotide, and 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). For example, when the polymer molecule is a protein, the structural unit is an amino acid, and the sequence of the structural unit is a sequence order of 20 or more known amino acids.

Each step of the present invention will be described below by taking as an example the case where the aforementioned base body 10B is used.
[Step A1]
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.
In the base body 10 </ b> B, 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. Moreover, as a method of introducing more directly, a method of transporting the polymer molecule into the through-hole 1 using optical tweezers can be exemplified.

[Step A2]
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.
As 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 1, 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.
As a method of fixing at least a part of the polymer molecule to the inner wall of the through-hole 1, 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]
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.
As a method for introducing a conjugate that binds to the polymer molecule fixed to the substrate 10B into the through-hole 1, 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. As a result, the combined body can be introduced into the through hole 1. In the through-hole 1, the conjugate binds to the polymer molecule.

When the polymer molecule is DNA or RNA, the conjugate is preferably a labeled deoxynucleotide, and the signal is preferably generated by a DNA or RNA replication reaction by DNA polymerase or RNA polymerase. By using this configuration, the base sequence of the DNA or RNA can be analyzed by the optical observation.

Hereinafter, the case where the analysis target is DNA will be described. However, when the analysis target is RNA, the analysis can be performed in the same manner as in the case where the analysis target is DNA after being converted to cDNA by reverse transcriptase. In addition, when the analysis target is RNA, it is also possible to directly visualize the reverse transcription reaction using RNA as a template using labeled deoxyribonucleotide and reverse transcriptase, and to sequence in the same manner as in the case of DNA. Further, 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.

[Step A4]
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.
In the through-hole 1, 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. At this time, a plurality of polymerases may be bound to one DNA strand. Among the substrates, labeled dATP (deoxyadenosine triphosphate), labeled dGTP (deoxyguanosine triphosphate), labeled dCTP (deoxycytidine triphosphate), and labeled dTTP (deoxythymidine triphosphate), the nucleotide sequence of the DNA chain A complementary labeled deoxynucleotide (hereinafter referred to as dNTP) is sequentially bound to DNA, and a DNA replication reaction is carried out from a predetermined position of the DNA. When each dNTP binds to the DNA strand, each dNTP is hydrolyzed to release pyrophosphate. By fluorescently labeling this pyrophosphate in advance, a fluorescent signal can be detected for each hydrolysis in the DNA replication reaction.

Since 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. By using different fluorescent labels corresponding to each of the four types of dNTPs, 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.

If multiple polymerases bind to DNA and signals are detected at multiple locations in the observation area, add the position information of the signal on the two-dimensional plane to the type and order of the one-dimensional fluorescent signal of each signal. By linking each signal information two-dimensionally, the sequencing speed can be dramatically accelerated.
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. By simultaneously performing the above reaction in a plurality of through-holes, it is possible to make the obtained data redundant and improve the sequencing accuracy.
As a method of connecting each signal information in a two-dimensional manner using the FIONA method, for example, the FIONA method in a motor protein described in the following reference is used, and in the present invention, the color of the fluorescent signal that appears sequentially This is applied to specify the position, and the position information is recorded in two-dimensional coordinates. Accordingly, there is a method of arranging each signal on the two-dimensional coordinate and constructing a DNA sequence to be analyzed on the two-dimensional coordinate.
(Reference; “Fluorescence Imaging with OneNometer Accuracy: Application to Molecular Motors” Ahmet Yildiz and Paul R. Servin, Acc. Chem. Res. 2005, 38. 58.

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. For example, a high-resolution CCD camera used in combination with an optical microscope can be used.

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. This is repeated for the type of amino acid determining molecule, and the entire sequence of the polypeptide is determined by connecting the obtained signal position and the type of amino acid determining molecule. By simultaneously performing the above reaction in a plurality of through-holes, it is possible to make the obtained data redundant and improve the sequencing accuracy.
Similar methods can be applied to DNA methylation and histone modifications, which are epigenetic landmarks in DNA. That is, after DNA sequencing, a fluorescent antibody that recognizes methylated DNA is introduced and bound to the methylated DNA. Thereafter, the position information of the fluorescent antibody is acquired by the same two-dimensional position determination as described above. Next, it is compared with the position information of the base sequence of DNA acquired previously, and which base is methylated is determined. Histone acetylation can also be detected by the same method. The method described above can be used for fixing the DNA in the channel.

Examples of the method for producing the “antibody recognizing 3 to 4 amino acids” include the following methods. When there are 20 types of amino acids constituting the polypeptide to be analyzed, 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. At this time, 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. . Next, the C-terminus of the synthesized peptide is immobilized on latex beads via an appropriate linker such as PEG (polyethylene glycol). Using these beads, an animal such as a mouse or a rabbit is immunized by a conventional method to obtain a polyclonal antibody. Note that 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.
By this method, 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. In the same manner, 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. Use each lot independently or simultaneously for the polypeptide to be analyzed, measure the position where the antibody corresponding to each amino acid type binds to the polypeptide, and determine the amino acid type at a specific position in the polypeptide . By integrating the obtained positional information and the type of amino acid, the amino acid sequence of the polypeptide can be determined.

<Method for producing substrate>
Next, a method for manufacturing a substrate according to the present invention will be described using the substrate 10A of the first embodiment as an example. As shown in FIGS. 15A to 15D, the laser L having a pulse time width of picosecond order or less (10 ps or less) is irradiated on the region to be the through hole 1 in the single member 4 to thereby change the region. Step M1 (FIG. 15A) for forming the mass portion 51, Step M2 (FIG. 15B) for forming the first flow path 2 and the second flow path 3 forming the space in the single member 4, and a single member 4 and at least a step M3 (FIG. 15C) of removing the modified portion 51 by etching.

[Step M1]
As the laser L (laser light L), it is preferable to use 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. However, it is necessary to use a wavelength that transmits the member 4. More specifically, a laser beam having a transmittance of 60% or more with respect to the member 4 is preferable.

As the laser L (laser light L), light in a general wavelength region (0.1 to 10 μm) used as a processing laser can be applied. Among these, it is necessary to penetrate the member 4 which is a workpiece. By applying laser light having a wavelength that passes through the member 4, 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. In particular, the material is preferably amorphous so as not to be affected by processing anisotropy due to crystal orientation.
Furthermore, when observing with optical means such as a microscope, it is more preferable to use glass, quartz, or sapphire because visible light (wavelength: 0.36 μm to 0.83 μm) is transmitted.

Further, it is preferable that the material of the member 4 transmits light having at least some wavelengths among light having wavelengths of 0.1 μm to 10 μm.
Specifically, it is preferable to transmit at least a part of light in a general wavelength region (0.1 μm to 10 μm) used as a processing laser beam. When the material of the member 4 transmits such laser light, the modified portion can be formed by irradiating the member with laser as described later.
More preferably, the material of the member 4 is transparent to light in the visible light region (wavelength of about 0.36 μm to about 0.83 μm). When 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.
In the present invention, “transmission (transparent)” refers to all states in which light enters the member and transmitted light is obtained from the member.
In FIGS. 15A to 15D, the single member 4 is a transparent glass substrate (hereinafter referred to as a glass substrate 4).
Below, although the case where the member 4 is a glass substrate is demonstrated, even if the member 4 is the case of another member, for example, a silicon | silicone, quartz, or sapphire, the same process can be performed.
Silicon, quartz, and glass are more suitable for the workability in the process M2 to be described later.

As 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. Further, the thickness of the glass substrate 4 is not particularly limited.

As the irradiation method of the laser beam L, the method shown in FIG. That is, 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.
By scanning the focal point inside the glass substrate 4 in the region to be the through hole 1, the modified portion 51 having a desired shape can be formed.
Here, in the present specification and claims, the “modified portion” means a portion having low etching resistance and selectively or preferentially removed by etching.

When irradiating the laser beam L, 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. Hereinafter, 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, and the polarization direction (electric field direction) of the laser light L is an arrow Y. In the laser irradiation method S, 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. At the same time, 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.

By this laser irradiation method S, the modified portion 51 having a nano-order aperture can be formed in the glass substrate 4. For example, 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 μm to 5 μm is obtained. In this substantially elliptical shape, the direction along the laser propagation direction is the major axis, and the direction along the laser electric field direction is the minor axis. Depending on the laser irradiation conditions, the cross section may have a shape close to a rectangle.

When the laser irradiation intensity is set to be equal to or higher than the processing upper limit threshold of the glass substrate 4, the obtained modified portion 51 may be formed with a periodic structure. In other words, by condensing and irradiating a pulse laser of picosecond order or less above the processing upper threshold, interference between the electron plasma wave and incident light occurs at the condensing part, and it is perpendicular to the laser polarization. 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. For example, in the case of quartz, 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.

On the other hand, as in the laser irradiation method S described above, if 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.

According to the laser irradiation method S described above, the shape of the cross section perpendicular to the longitudinal direction of the through-hole 1 can be an ellipse or a substantially ellipse. In addition, the minor axis can be controlled to a nano-order size by etching. In the case of an ellipse or a substantially elliptical shape, 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. Moreover, 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. In this case, since 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. However, by making the cross-section of the through-hole 1 elliptical or substantially elliptical, the major axis is made sufficiently large even if the polymer molecule is introduced or the minor axis is sufficient to capture the microorganism. Thus, 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.

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.

Therefore, the processing upper limit threshold (processing appropriate value) 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.

[About laser irradiation intensity]
In the present specification and claims, 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.
In the present specification and claims, 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.

Although the above-described polarization has been described in detail with respect to linear polarization, it can be easily imagined that a similar structure (modified part) is formed even with a laser pulse having some elliptical polarization component.

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.

In FIGS. 17A and 17B, 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.
When 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. 4, in order to form the modified portion 51 in which the direction of the major axis is inclined with respect to the main surface 4a, 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).

Generally, 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.

It is also possible to adjust the shape of the modified portion formed in the glass substrate 4 in the three-dimensional direction by appropriately changing the laser polarization direction (arrow Y direction).

Further, as shown in FIG. 17A, the modified portion 51 may be formed by condensing the laser light L with a lens and irradiating it as described above.
As the 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. Further, for example, if a cylindrical lens is used, it is possible to irradiate a wide area of the glass substrate 4 with a laser at a time. For example, if a conical lens is used, the laser beam L can be irradiated at once in a wide range in the vertical direction of the glass substrate 4. However, when a cylindrical lens is used, the polarization of the laser light L needs to be horizontal with respect to the direction in which the lens has a curvature.

Specific examples of the laser irradiation condition S include the following various conditions. For example, a titanium sapphire laser (a laser using a crystal in which sapphire is doped with titanium as a laser medium) is used. As 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.

As a lens used for condensing, for example, N.I. A. It is preferable to use an objective lens of <0.7. In order to form a finer through-hole 1, 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.
First, a resist 52 is patterned and arranged on the upper surface of the glass substrate 4 by, for example, photolithography (FIG. 15B).
Next, 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).
Finally, when the resist 52 that has become unnecessary is removed, the glass substrate 4 on which the first flow path 2 and the second flow path 3 are formed is obtained.

In 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]
Next, the modified portion 51 formed in step M1 is removed from the single glass substrate 4 by etching (FIG. 15D).
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.

As a result of the etching, 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 μm to 5 μm. In addition, depending on the degree of etching, the cross section may be a shape close to a rectangle.

By adjusting the processing time of the wet etching, 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 μm to 2 μm and the major axis can be set to about 5 μm to 10 μm.

Next, 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.

As the etching in the process M2 and the process M3, wet etching or dry etching can be applied. For wet etching, it is most preferable to use, for example, 1% or less hydrofluoric acid, but other acid or basic containers may be used.

Among the dry etching methods, 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.

As an 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). For example, it is possible to use etching using a neutral particle beam. When the anisotropic dry etching method is used, 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.

In step M2, a more preferable etching is anisotropic etching, and in step M3, a more preferable etching is isotropic etching.

DESCRIPTION OF SYMBOLS 1 ... Through-hole, 2 ... 1st flow path (space), 2a ... Side surface of 1st flow path, 3 ... Second flow path (space), 3a ... Side surface of 2nd flow path, 4 ... Base material, 4a ... Base material 5 ... a member to be a lid, 6 ... a vertical hole, 7 ... a third channel, 8 ... a fourth channel, 9 ... a landing, 10A to 10H (10) ... a base, 11 ... Temperature control device, 51 ... reforming section, 52 ... resist, L ... laser beam

Claims

A substrate,
A base material provided with a space into which a solution containing polymer-like molecules flows, and
A through-hole having a shape that is formed inside the base material, is open to the space, and is capable of extending and arranging the polymer-like molecule therein;
With
The base | substrate characterized by the site | part which comprises the said through-hole at least among the said base materials being formed with a single member.
The substrate according to claim 1,
The through hole is at least partially columnar.
The substrate according to claim 1 or 2,
A substrate having a length in the longitudinal direction of the through hole of 0.1 μm to 10 mm.
A substrate according to any one of claims 1 to 3,
A substrate having a minor axis of a cross section perpendicular to the longitudinal direction of the through hole of 1 nm to 1000 nm.
A substrate according to any one of claims 1 to 4,
The base material is a substrate having a main surface, the cross-sectional shape orthogonal to the longitudinal direction of the through hole is substantially elliptical, and the major axis of the elliptical shape is inclined with respect to the main surface. A substrate characterized by comprising:
A substrate according to any one of claims 1 to 5,
The substrate further comprising a vertical hole communicating the through hole and the outside of the substrate.
A substrate according to any one of claims 1 to 6,
A substrate having a fixing part for fixing at least a part of the polymer molecule inside the through-hole.
The substrate according to claim 7,
The base body, wherein the fixing portion is made of metal.
A substrate according to any one of claims 1 to 8,
A substrate characterized in that the polymer molecule is DNA, RNA, or polypeptide.
An analysis method,
A base material provided with a space into which a solution containing polymer-like molecules flows is formed, and is formed inside the base material and is open to the space, and the polymer-like molecules are stretched and arranged inside the base material. A through hole having a shape that can be formed, and at least a portion of the base material forming the through hole is formed of a single member. Using the substrate according to one item,
Flowing a first solution containing the polymer-like molecule into the space, introducing the polymer-like molecule into the through-hole,
Fixing at least a part of the polymeric molecule to the inner wall of the through-hole,
Introducing a conjugate that binds to the polymeric molecule into the through-hole,
An analysis method, wherein a signal generated as a result of the binding is optically observed from the outside of the substrate.
The analysis method according to claim 10, comprising:
An analysis method, wherein the conjugate is bound to each of a plurality of locations in the polymer-like molecule, and a plurality of signals generated as a result of the binding are detected in association with positional information of the plurality of locations.
The analysis method according to claim 10 or 11,
The polymer molecule is DNA or RNA, the conjugate is a labeled deoxynucleotide, the signal is generated by a replication reaction of DNA or RNA by a polymerase, and the base sequence of the DNA or RNA is determined by the optical observation. An analysis method characterized by analyzing.
The analysis method according to any one of claims 10 to 12,
An analysis method, wherein the second solution is circulated in the through hole.