JP2004317340A - Manufacturing method of columnar structure, and electrophoretic device using columnar structure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a manufacturing method of a columnar structure capable of acquiring a desired micro-aperture, and an electrophoretic device capable of accurately separating DNA or the like. <P>SOLUTION: Many pillars 3 are formed on a silicon substrate 2 by ICP-RIE, and the pillars 3 are thermally oxidized, and a polycrystal silicon film is stuck on the thermally-oxidized pillars 3. The polycrystal silicon film is thermally oxidized, and a polycrystal silicon film 12 is stuck onto the thermally-oxidized polycrystal silicon film. This electrophoretic device has a micro-passage constituted of the columnar structure manufactured by the measuring method and a glass substrate. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for manufacturing a columnar structure in which a large number of columnar projections are provided on a silicon substrate, and an electrophoretic device having the columnar structure.
[0002]
[Prior art]
2. Description of the Related Art In recent years, attention has been focused on analysis techniques and separation techniques for DNA and RNA, which are one field of biotechnology. Conventionally, as a DNA separation means, an electrophoresis method using a mesh gel or a polymer has been known.
[0003]
Recently, a technique has been reported in which a columnar structure in which fine columns are erected on a silicon substrate is used instead of a gel or a polymer (for example, see Patent Document 1).
This silicon columnar structure is manufactured by a method of expanding the volume of the silicon column by thermal oxidation and narrowing the gap between the columns. As a result of the thermal oxidation, the entire column is made of silicon oxide or covered with silicon oxide. The gap between the columns works like a gel or polymer network.
Further, there has been reported a device in which a cover glass is arranged on the columnar structure to form a concave flow path and the DNA in the concave flow path is separated by electrophoresis.
[0004]
[Patent Document 1]
JP-A-4-21232 (pages 4, 5; FIGS. 7, 8)
[0005]
[Problems to be solved by the invention]
The above-described method for manufacturing a silicon columnar structure has a problem that it is difficult to control a minute gap because a single thermal oxidation narrows the gap between silicon columns. That is, a desired gap cannot be obtained unless the etching conditions for forming the silicon pillars and the thermal oxidation conditions for expanding the silicon pillars are strictly controlled.
[0006]
The present invention provides a method for producing a columnar structure capable of obtaining a desired fine gap, and an electrophoresis device capable of accurately separating DNA and the like.
[0007]
[Means for Solving the Problems]
(1) A method for manufacturing a columnar structure according to claim 1 includes a step of forming a large number of columnar projections (pillars) on a silicon substrate by etching, a step of thermally oxidizing the columnar projections, and a step of thermally oxidizing the columnar projections. The method includes a step of attaching a polycrystalline silicon film, a step of thermally oxidizing the polycrystalline silicon film, and a step of attaching a polycrystalline silicon film to the thermally oxidized polycrystalline silicon film.
[0008]
(2) In the method for manufacturing a columnar structure according to claim 1, it is preferable that the step of attaching a polycrystalline silicon film to the columnar protrusions thermally oxidized and the step of thermally oxidizing the polycrystalline silicon film be repeated.
(3) In the method for manufacturing a columnar structure, the etching method is preferably ICP-RIE, and the polycrystalline silicon film is preferably deposited by LPCVD.
[0009]
(4) A columnar structure according to a fifth aspect is a columnar structure manufactured by any one of the above-described manufacturing methods, wherein the surface has a polycrystalline silicon film.
[0010]
(5) The electrophoretic device according to claim 6 has a microchannel formed by joining a transparent substrate to the tip of the columnar protrusion of the columnar structure according to claim 5, and the electrophoresis device causes the microchannel in the microchannel by electrophoresis. The sample is passed and separated.
(6) In the above electrophoretic device, it is preferable that the columnar structure and the transparent substrate are bonded by an anodic bonding method.
(7) It is preferable that the electrophoresis device further includes a storage unit that stores a sample to be supplied to the microchannel and a sample to be collected from the microchannel.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
(First Embodiment)-Manufacturing method of columnar structure-
Hereinafter, a method for manufacturing a columnar structure according to the present invention will be described with reference to the drawings.
FIG. 1 is a partial perspective view schematically showing the structure of the columnar structure according to the first embodiment of the present invention. FIG. 2 is a partial cross-sectional view showing a manufacturing process of the columnar structure of FIG.
[0012]
In FIG. 1, a columnar structure 1 includes a silicon substrate 2 and columnar projections (pillars) 3, and the silicon substrate 2 and the pillar 3 have an integral structure. A pillar array 4 in which a number of pillars 3 are arranged is formed in a concave portion 5 of the silicon substrate 2. The height of the pillar 3 is usually equal to the depth of the recess 5. In addition, the arrangement pitch of the pillars 3 and the gap between the pillars 3 are often constant, but may differ depending on the location as described later.
[0013]
From the viewpoint of the material, the silicon substrate 2 includes a silicon base material 2b, a silicon oxide layer 11, and a polycrystalline silicon film 12 in this order from the inside toward the surface. The pillar 3 has a material configuration in which the inside is a silicon oxide layer 11 and the polycrystalline silicon film 12 is formed outside the silicon oxide layer 11. However, when the pillar 3 is thick, the material may have a structure in which silicon is present at the center, similarly to the silicon substrate 2. In any case, the substrate surface 2a, the bottom surface of the concave portion 5, the side surface of the concave portion 5, and the surface 3a of the pillar 3 are all polycrystalline silicon films 12.
[0014]
Here, the manufacturing process of the columnar structure 1 will be described with reference to FIG. FIG. 2 is a partial cross-sectional view for explaining a manufacturing procedure of the columnar structure, and is a view cut along a plane passing through the central axis of each pillar. The manufacturing process proceeds in order from (a) to (f). Since the material and volume of the pillar 3 change as the process progresses, the pillar in each process is represented by reference numerals 3b to 3f.
[0015]
FIG. 2A shows a state in which the shape to be the pillar array 4 is patterned on the photoresist film 100 applied on the silicon substrate 2.
FIG. 2B shows a state of a pillar when the silicon substrate 2 is etched by ICP-RIE (inductively coupled plasma-reactive ion etching). For example, when the etching depth is 10 μm, the diameter of each pillar 3b is 2 μm, the height of each pillar 3b is 10 μm, and the interval between the pillars 3b, that is, the arrangement pitch is 4 μm.
[0016]
In the ICP-RIE, a sample is etched under a relatively low pressure of 0.05 to 1 Pa using a chemical reaction between ions of a process gas in a high-density plasma and the sample surface. High etching process is possible. An oxidizing gas such as CCl ₂ F ₂ or CF ₄ is used as the process gas.
[0017]
FIG. 2C shows a state of the pillar 3c on which the silicon oxide layer 11 is formed by thermal oxidation. Examples of the thermal oxidation include dry oxidation using O ₂ gas and steam oxidation using steam or a mixed gas of H ₂ O and O ₂ . When thermal oxidation is performed at 1050 ° C. for 10 hours, the silicon oxide has a thickness of 2 μm, so that all the pillars 3c become silicon oxide and volume expansion occurs. The silicon oxide may be formed by an oxidation method other than thermal oxidation, for example, plasma oxidation for ionizing O ₂ gas or anodic oxidation using an ethylene glycol solution.
[0018]
FIG. 2D shows a state of a pillar 3d in which a polycrystalline silicon film 12a is deposited to a thickness of 200 nm by LPCVD (low pressure chemical vapor deposition).
LPCVD is a film formation method in which a sample is heated under a reduced pressure of 10 to 10 ³ Pa, and a film is formed on the sample surface by a gas phase chemical reaction using thermal energy. This method has an advantage that the film can be deposited easily and a uniform film thickness can be obtained. In forming polycrystalline silicon, SiCl ₄ + H ₂ or SiH ₄ is used as a process gas.
[0019]
FIG. 2E shows a state of the pillar 3e in which the polycrystalline silicon film 12a is oxidized by the second thermal oxidation at 1050 ° C. for 2 hours and the silicon oxide layer 11 is additionally formed.
[0020]
FIG. 2F shows a state of the pillar 3f in which the polycrystalline silicon film 12 is deposited on the surface of the pillar 3e of FIG. This state is the same as the state shown in FIG.
[0021]
Next, adjustment of the gap between the pillars 3 will be described. The gap is accurately adjusted by controlling the volume expansion of the pillar 3 due to thermal oxidation and controlling the film thickness of the polycrystalline silicon films 12 and 12a at the time of film formation.
According to the theory of thermal oxidation, 44% of the volume of silicon oxide corresponds to the volume of silicon before oxidation. Therefore, if the radius of the pillar 3c after the thermal oxidation is Rc and the radius of the pillar 3b before the thermal oxidation is Rb, there is a relationship of Expression 1.
(Equation 1)
Rc ³ = Rb ³ /0.44 (1)
However, according to the thermal oxidation experiment, the relationship of Expression 2 is established. Therefore, in this embodiment, Expression 2 is used.
(Equation 2)
Rc ³ = Rb ³ /0.55 (2)
[0022]
When the polycrystalline silicon film is thermally oxidized, the polycrystalline silicon film is extremely thin, so that a planar approximation holds with respect to Equation (1). That is, assuming that the thickness of the polycrystalline silicon film before thermal oxidation is t1 and the thickness after thermal oxidation is t2, the relationship of Equation 3 holds. Equation 3 has been confirmed to be compatible with experimental values.
[Equation 3]
t2 = t1 / 0.44 (3)
[0023]
If Equations 2 and 3 are applied to the setting of the thermal oxidation conditions, the volume expansion of the pillar 3 can be controlled. Also, the amount of increase in the diameter of the pillar 3 can be controlled by controlling the thickness of the polycrystalline silicon using LPCVD. In polycrystalline silicon film formation using LPCVD, since the film thickness can be controlled with an accuracy of 1 nm, the gap between the pillars 3 can be controlled with an accuracy of 1 nm.
[0024]
For example, in FIGS. 2B and 2C, since the diameter of the pillar 3b is 2 μm, if thermal oxidation is performed completely, the diameter of the pillar 3c is 2.44 μm from Equation 2. 2D and 2E, the diameter of the pillar 3d on which the polycrystalline silicon film 12a is deposited to a thickness of 200 nm is 2.84 μm. .35 μm. The polycrystalline silicon film 12a having a thickness of 200 nm can be completely oxidized under a thermal oxidation condition of 1050 ° C. × 1.5 hours. However, in order to achieve complete oxidation, a thermal oxidation of 1050 ° C. × 2 hours is performed. You have selected a condition.
[0025]
However, when the polycrystalline silicon film is formed thick at a time, the accuracy of controlling the film thickness is reduced. Also, during thermal oxidation, the oxidation rate decreases as the inside of the film is increased. Therefore, when the polycrystalline silicon film is formed to be relatively thin and thermal oxidation is performed, the film thickness accuracy is improved and the oxidation rate is increased. By repeating the polycrystalline silicon film formation and the thermal oxidation, the accuracy of the gap G between the pillars 3 is improved, and the efficiency of the entire process is increased.
[0026]
By repeating the steps of FIG. 2D and FIG. 2E, for example, if a second oxidation is performed after forming a polycrystalline silicon film having a thickness of 80 nm, the diameter of the pillar 3e can be reduced from 3.35 μm. Increase to 3.71 μm. In the step (b), the pitch between the pillars 3b is 4 μm, so that the gap between the pillars 3e is 0.29 μm.
[0027]
In the step of FIG. 2F, a polycrystalline silicon film 12 is formed on the outermost surface of the pillar 3f. This polycrystalline silicon film 12 is not subjected to thermal oxidation. For example, if the thickness of the polycrystalline silicon film 12 is 50 nm, the diameter of the pillar 3f is 3.81 μm, and the gap G between the pillars 3f is 0.19 μm.
[0028]
By manufacturing the columnar structure 1 using the manufacturing method as described above, the final gap G between the pillars 3 can be formed with an accuracy of 1 nm.
[0029]
(Second embodiment)-Electrophoresis device-
FIG. 3 is a plan view schematically showing the entire configuration of the electrophoresis device according to the second embodiment of the present invention. On the silicon substrate of this electrophoretic device, the columnar structures according to the first embodiment are formed with the same manufacturing method and the same dimensions. Hereinafter, the reference numerals of the components of the columnar structure 1A are the same as those of the columnar structure 1 in FIG.
FIG. 4 is a partial perspective view schematically showing a microchannel formed by bonding a silicon substrate and a glass substrate. In FIG. 4, only the area where the columnar structure 1A exists is shown, and the pillar array 4 standing in the recess 5 is not shown.
[0030]
As shown in FIG. 3, the electrophoresis device 20 includes a silicon substrate 2A provided with a columnar structure 1A, reservoirs (storage portions) 21 to 24, channels 25 and 26, and electrodes 27a, 27b, 28a, and 28b, and glass. The substrate 30 is configured to be bonded. FIG. 3 corresponds to a view in which the silicon substrate 2A is seen through the glass substrate 30.
Pyrex glass (registered trademark of Corning Incorporated, USA) having a thickness of 50 to 500 μm is used as the glass substrate. Further, glass containing an alkali metal such as Li, Na, and K can also be used.
[0031]
The four reservoirs 21 to 24 are recesses for storing samples of DNA molecules, RNA molecules, and the like supplied from the outside, and for collecting samples separated by electrophoresis. The channels 25 and 26 are grooves having a width of, for example, 50 μm and communicating with each other at the intersection C. Channel 25 is connected to reservoirs 22 and 24, and channel 26 is connected to reservoirs 21 and 23.
[0032]
The columnar structure 1A constitutes a part of the channel 26 of the electrophoresis device 20. The columnar structure 1 </ b> A is disposed at a position about 0.1 to 0.3 mm away from the intersection C toward the reservoir 23. The length of the columnar structure 1A along the channel is set based on the migration distance that can separate DNA molecules and the like, and is, for example, in the range of 0.3 to 50 mm.
The columnar structure 1A, the reservoirs 21 to 24 and the channels 25 and 26 are formed integrally with the silicon substrate 2A by a silicon micromachining technique, that is, silicon micromachining.
[0033]
The electrodes 27a, 27b, 28a, 28b are formed on the silicon substrate 2A by depositing a conductive film such as Au, Pt, or the like by using evaporation or sputtering. The electrode 27a is arranged near the reservoir 22 and the electrode 27b is arranged near the reservoir 24. The electrode 28a is arranged near the reservoir 21 and the electrode 28b is arranged near the reservoir 23.
[0034]
In FIG. 3, the electrodes 27a and 27b are respectively connected to the negative electrode and the positive electrode of the DC power supply 27c. The electrodes 28a and 28b are connected to a negative electrode and a positive electrode of the DC power supply 28c, respectively. However, as described later, when performing the electrophoresis process, the negative electrode is switched to ground, and the positive electrode is switched to ground as needed.
[0035]
The silicon substrate 2A is bonded to a glass substrate by anodic bonding after the columnar structure 1A, the reservoirs 21 to 24, the channels 25 and 26, and the electrodes 27a, 27b, 28a and 28b are provided. Prior to the anodic bonding, four through holes (not shown) positioned so as to communicate with the reservoirs 21 to 24 are formed in the glass substrate. The diameter of the through hole is 1 to 3 mm. Ultrasonic processing, abrasive grain injection processing, or the like is used to form the through holes.
[0036]
In FIG. 4, only the area where the columnar structure 1A exists is shown, and the pillar array 4 standing in the recess 5 is not shown. As shown in the figure, the upper part of the concave portion 5 of the columnar structure 1A is closed by the glass substrate 30 to form a tunnel-shaped microchannel 10 extending in the direction of the arrow.
[0037]
The anodic bonding for bonding the silicon substrate 2A and the glass substrate 30 will be described.
The surface 2a of the silicon substrate 2A and the surface 3a of the pillar 3 are both polycrystalline silicon films 12. As shown in the figure, a high electric field of 500 to 800 V is applied while the columnar structure 1A is in contact with the glass substrate 30 and heated to 500 to 600 ° C. At this time, assuming that the columnar structure 1A is an anode and the glass substrate 30 is a cathode, at the interface between the two, a positive charge is accumulated on the columnar structure 1A side and a negative charge is accumulated on the glass substrate 30 side. Due to the electrostatic attraction acting between the positive charge and the negative charge, Si atoms in the polycrystalline silicon film 12 and oxygen ions in the glass substrate 30 are chemically bonded. That is, the polycrystalline silicon film 12 on the outermost surface of the columnar structure 1A functions as an adhesive, and the surface 2a of the silicon substrate 2A and the head of each pillar 3 are firmly joined to the glass substrate 30.
[0038]
Therefore, when a sample such as a DNA molecule or an RNA molecule is flown through the microchannel 10, there is no possibility that the sample leaks outside. Similarly, there is no possibility that the sample leaks from the reservoirs 21 to 24 and the channels 25 and 26 shown in FIG.
[0039]
In the conventional DNA separation device, the cover glass is arranged on the pillar. However, since the pillar is made of silicon oxide, it is difficult to obtain a sufficient bond between the pillar and the cover glass. there were. If sufficient binding is not ensured, a sample such as DNA in the flow path may overflow during the separation operation, which may hinder accurate data acquisition and observation of the separation process.
[0040]
As described in the first embodiment, the polycrystalline silicon film 12 has a very small thickness of 50 nm, and thus has a high resistance. The entire inner surface of the microchannel 10 is covered with the polycrystalline silicon film 12. Therefore, even if the electrodes 27a, 27b, 28a, and 28b are formed directly on the polycrystalline silicon film 12 and an electric field is applied, problems such as a voltage drop and a short circuit caused by a current flowing through the polycrystalline silicon film 12 do not occur.
[0041]
Next, a procedure for separating DNA using the electrophoresis device 20 of the present embodiment will be described with reference to FIG.
After the analysis buffer solution is injected into the electrophoresis device 20, the DNA sample stained with the fluorescent reagent is supplied to the reservoir 22. DNA samples are mixed samples having various molecular sizes. A voltage is applied using the electrode 27b as a positive electrode. At this time, the electrodes 27a, 28a, 28b are grounded. Since the DNA molecule is negatively charged, the DNA molecule moves from bottom to top on the paper surface in the channel 25.
[0042]
When the DNA sample moves to the intersection C, a voltage is applied with the electrode 28b as the positive electrode. At this time, the electrodes 27a, 27b, 28a are grounded. The DNA sample changes its direction and moves from right to left on the paper surface in the channel 26, passes through the microchannel 10, and is collected by the reservoir 23.
[0043]
As an improved example of the electrophoretic device, there is a configuration in which an AC circuit or an electromagnetic coil is added to the electrophoretic device of FIG. In FIG. 3, an AC electric field is applied to the columnar structure 1A in the width direction (vertical direction on the paper) by an AC circuit (not shown). By this action, the DNA molecules migrate while being elongated in the width direction. Since a moment is applied to the DNA molecule, a difference occurs in the migration speed according to the molecular length. Each DNA molecule has this velocity difference, resulting in more precise separation. The AC voltage is in the range of several volts to 1000 volts.
In FIG. 3, when a magnetic field is generated in a vertical direction (a direction perpendicular to the paper surface) of the columnar structure 1A by an electromagnetic coil (not shown), more precise separation of each DNA molecule is performed by the same operation. Done. The strength of the magnetic field ranges from 0.1 to 10 Tesla.
[0044]
The appearance of the DNA sample passing through the microchannel 10 can be observed through the glass substrate 30 with a fluorescence microscope. The objective lens of the fluorescence microscope is set right above the microchannel 10 so that the optical axis is orthogonal to the surface of the glass substrate 30. Although the thickness of the glass substrate 30 is 50 to 500 μm, it is desirable to use a thin glass substrate when the observation magnification is high because the depth of focus is short.
[0045]
In the present invention, a silicone rubber sheet, a synthetic rubber sheet, or the like can be used as a transparent substrate other than the glass substrate. These rubber sheets are joined by being pressed against the surface 2a of the silicon substrate 2A and the surface 3a of the pillar 3.
[0046]
Electrophoresis was performed using a DNA sample having a molecular size of 200 bp and 300 bp, with the length of the microchannel 10 set to 1 mm, the gap between the pillars set to 300 nm, and the applied voltage between the electrodes 28a and 28b set to 600V. In the observation by the fluorescence microscope, a state where the DNA molecules were moving in the microchannel 10 could be observed. In addition, separation of two types of DNA molecules was confirmed.
[0047]
In the electrophoresis device 20 of the present embodiment, the gap between the pillars standing in the microchannel 10 is 300 nm. However, the present invention is not limited to this, and is appropriately selected according to the molecular size of the DNA sample. be able to. When the molecular size is large, the gap between the pillars is widened, and when the molecular size is small, the gap between the pillars is narrowed, thereby enabling accurate separation in a short time. To adjust the gap between the pillars, the thickness of the polycrystalline silicon films 12 and 12a may be controlled as described in the first embodiment.
[0048]
Although the arrangement pitch of the pillar array 4 is constant at 4 μm, the arrangement pitch may be increased in the upstream portion of the microchannel 10 and reduced in the downstream portion. The arrangement pitch may be changed continuously. This can be realized by the patterning of FIG. Thereby, the gap between the pillars also changes. For a DNA sample having a wide range of molecular sizes, clogging of large molecular size DNA can be prevented, and small molecular size DNA can be accurately separated.
[0049]
Further, the shape of the pillar 3 is a straight cylinder, but may be formed so that the diameter of the pillar changes in the height direction, or the shape of the pillar may be partially bent. A portion having a large gap between pillars has an effect of preventing clogging of DNA having a large molecular size, and a portion having a small gap between pillars can accurately separate DNA having a small molecular size.
[0050]
The columnar structure according to the production method of the present invention can be applied not only to an electrophoretic device but also to various filters such as a protein separation filter and a fine particle filter.
[0051]
【The invention's effect】
As described above, if a columnar structure is manufactured by the manufacturing method of the present invention, a desired fine gap can be obtained. The electrophoresis device having the columnar structure enables accurate separation of DNA and the like.
[Brief description of the drawings]
FIG. 1 is a partial perspective view schematically showing a structure of a columnar structure manufactured by a manufacturing method according to a first embodiment of the present invention.
FIG. 2 is a partial cross-sectional view showing a manufacturing process of the columnar structure according to the first embodiment of the present invention.
FIG. 3 is a plan view schematically showing an overall configuration of an electrophoretic device according to a second embodiment of the present invention.
FIG. 4 is a partial perspective view schematically showing a microchannel as a component of an electrophoresis device according to a second embodiment of the present invention.
[Explanation of symbols]
1, 1A: columnar structure 2, 2A: silicon substrate 3: pillar (columnar projection)
4: pillar array 5: recess 10: microchannel 11: silicon oxide layers 12, 12a: polycrystalline silicon film 20: electrophoretic devices 21 to 24: reservoirs 25, 26: channels 27a, 27b, 28a, 28b: electrodes 30 ： Glass substrate

Claims (8)

Forming a large number of columnar projections by etching on the silicon substrate;
Thermally oxidizing the columnar protrusions;
Attaching a polycrystalline silicon film to the thermally oxidized columnar protrusions,
Thermally oxidizing the polycrystalline silicon film;
Attaching a polycrystalline silicon film to the thermally oxidized polycrystalline silicon film. The method for manufacturing a columnar structure according to claim 1,
A method of manufacturing a columnar structure, comprising repeating the step of attaching a polycrystalline silicon film to the thermally oxidized columnar projections and the step of thermally oxidizing the polycrystalline silicon film. The method for manufacturing a columnar structure according to claim 1 or 2,
The method for manufacturing a columnar structure, wherein the etching method is ICP-RIE. The method for manufacturing a columnar structure according to claim 1 or 2,
The method of manufacturing a columnar structure, wherein the method of attaching the polycrystalline silicon film is LPCVD. A columnar structure manufactured by the manufacturing method according to claim 1, wherein a surface of the columnar structure has a polycrystalline silicon film. 6. A column having a micro channel formed by joining a transparent substrate to a tip of a columnar projection of the columnar structure according to claim 5, wherein a sample is passed through the micro channel by electrophoresis and separated. Electrophoresis device. The electrophoretic device of claim 6,
An electrophoretic device, wherein the columnar structure and the transparent substrate are bonded by an anodic bonding method. The electrophoretic device of claim 6,
An electrophoresis device further comprising a storage unit for storing a sample to be supplied to the microchannel and a sample to be collected from the microchannel.

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