JP2003098146A - Apparatus for recognizing base sequence, its manufacturing method and its using method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus for determining the base sequence of DNA inexpensively, efficiently and quickly. SOLUTION: The apparatus is manufactured using semiconductor micromachining technology and comprises two liquid reservoirs separated by an insulation film having pores, two electrodes disposed in two liquid reservoirs, respectively, and a device for measuring a metal ion current passing through the pores when a voltage is applied between the two electrodes.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to fine arrays, especially DN.
The present invention relates to a highly efficient fine sequence recognition device for A base sequences, and more particularly to a fine sequence recognition device for DNA using small holes produced by semiconductor fine processing technology.

[0002]

2. Description of the Related Art Deoxyribonucleic acid (DNA) nucleotide sequencing and protein amino acid sequencing are highly reliable.
Low cost and high speed are desired.

As a method for determining the nucleotide sequence of DNA, a chemical method (Proceedings of National Academy of Science, USA, Vol. 74, Vol. 5)
60 to 564, 1977) and enzymatic methods (Proceedings of National Academy of Sciences, USA, 74, 54).
Pp. 63 to 5467, 1977) was developed in the 1970s.

The chemical method is a method in which double-stranded DNA is dissociated into single strands and only one of four types of bases in single-stranded DNA is degraded. If the decomposition is gentle,
Only one base is decomposed at a random position per molecule of DNA. As a result, a group of DNA fragments having different lengths is obtained, and the difference in length is due to the decomposition of one of the four kinds of bases at different positions in the original DNA molecule. When the same operation is performed on all the bases and each DNA fragment group is subjected to gel electrophoresis in parallel, the base sequence can be read from the band pattern.

Due to the low base specificity and inaccuracies of chemical methods, enzymatic methods are the predominant method of sequencing today.

[0006] In the enzymatic method, first, DNA is synthesized in vitro using a DNA polymerase with a DNA whose sequence is to be determined as a template. At that time, four kinds of deoxyribonucleoside triphosphates which are synthetic raw materials and one small amount of dideoxyribonucleoside triphosphates are added. When the dideoxyribonucleoside triphosphate is incorporated into the DNA chain, the next nucleotide cannot be added. For example, excess deoxy ATP (dATP) and dideoxy ATP
When (ddATP) competes, the DNA strand synthesis by DNA polymerase randomly stops at A. Next, the base sequence is read by performing gel electrophoresis in the same manner as the chemical method.

The length of bases that can be sequenced by an enzymatic method is about several hundred. This is because it is limited by the resolution of gel electrophoresis. Therefore, it is necessary to fragment long DNA into short DNA. First, 1 DNA
Start by making a clone with a length of 0 kbase to 100 kbase to create a DNA library that covers all DNA. Each clone is further cleaved to form a DNA fragment having a length that can be analyzed, and the nucleotide sequence is determined. Finally, by reconstructing the base sequence-determined DNA fragment, the original complete base sequence of DNA is obtained. This method is widely used today because it is easy to operate and can be processed in parallel.

However, when creating a subclone, since there are many DNA fragments containing the same base sequence, a base length 10 to 20 times that of the portion for which the base sequence is to be determined is to be analyzed. On the contrary, if the overlapping portion is small, it becomes difficult to reconstruct the DNA fragment whose nucleotide sequence has been determined.

The primer walking method is a method that does not require repeated determination of the same base sequence many times (Science, vol. 258, pp. 1787 to 1).
791, 1992). In the primer walking method, huge DNA is used as it is as sample DNA. First, the base sequence of a part of the sample DNA is determined. Next, based on the determined base sequence, a primer that specifically hybridizes to the base sequence determined portion is synthesized to determine the DNA base sequence of the next portion. In the primer walking method, the portion where the base sequence is determined in duplicate can be minimized, but it is necessary to synthesize a primer for each base sequence determination.

Similarly, there is a channel method (US Paten) as a method for directly determining the base sequence of a huge DNA.
t 6015714 or Proceedings of National Academy of Sciences, USA, Vol. 93, pp. 13770 to 13773.
P., 1996). In the channel method, a solution containing metal ions is divided into two by a lipid bilayer having small pores (channels), and one solution is filled with single-stranded DNA. When a voltage is applied to the separated solution, the negatively charged DNA moves along the potential difference through the small pores to the other solution. Since the size of each of the four types of bases that compose DNA is different, the ratio of blocking the small pores differs depending on the difference in the bases. Therefore, when the ionic current in the small holes is measured, the ionic current differs due to the difference in base. By sequentially measuring the ionic current while DNA is passing through the small pores,
The base sequence of DNA can be determined.

For the small holes, alpha-hemolysin which is a toxin of Staphylococcus aureus which is a food poisoning bacterium, and maltoporin of Escherichia coli are used. Alpha-hemolysin and maltoporin, called channel proteins, form small pores with several nanometer openings in the lipid bilayer. In addition to DNA sequencing, channel proteins can be used for molecular counting using a molecular adapter (Nature, vol. 398, pages 686 to 690, 1999) and polymer counting (Nature, vol. 370, vol. 279 to 28
1 page, 1994), etc., and is known to work as a biosensor.

[0012]

In the base sequence determination by the chemical method and the enzymatic method, it is necessary to read the same base sequence portion in duplicate, and if the scale of the base sequence becomes large, it takes a lot of time and labor. . If the duplication is reduced to improve the efficiency, it becomes difficult to connect the respective DNA fragments whose base sequences have been determined, and there is a problem that the original long DNA base sequence cannot be reconstructed. In addition, there is a problem that a sequence that is difficult to be cloned exists, an unreadable portion occurs, and it cannot be reconstructed.

In the primer walking method, the next primer is always synthesized from the base sequence-determined portion and used, so that there is an advantage that the connection between the base sequence-determined portions is clear. However, there is a limit to the processing capacity because it is necessary to perform sequencing on one DNA strand from one side for every several hundred bases. Furthermore, a primer synthesized based on the sequenced portion works well. Without this, there was a problem that it became difficult to determine the entire base sequence.

Since the channel method uses alpha-hemolysin, which is a toxin of Staphylococcus aureus, which is a food poisoning bacterium, maltoporin of Escherichia coli, etc. in the pores, the pore formation itself is dangerous. Also, the formation of small holes is contingent,
It is difficult to control the number and position of channels formed in the lipid bilayer. For the above reasons, only principle experiments have been conducted so far, and nucleotide sequencing and molecular measurement have not been generally performed except for some biochemists.

[0015]

To solve the above problems, the object of the present invention is to eliminate the need for dividing DNA into fragment DNA,
It is to provide a high-speed and highly controllable base sequence determination device. Another object of the present invention is to provide a device that enables recognition of not only DNA but also minute sequences such as molecules and cells.

[0016]

Means for Solving the Problems D of the constitution (A) of the present invention
In the NA fine array recognition device, an insulating film having small holes, two liquid reservoirs separated by the insulating film and containing a solution containing metal ions, two electrodes installed in each of the two liquid reservoirs, and two liquid reservoirs It is equipped with a voltage source for applying a voltage between the electrodes and an ammeter capable of measuring the current due to the metal ions passing through the small holes.

In the DNA fine sequence recognition apparatus of the constitution (B) of the present invention, an insulating film having small holes, two liquid reservoirs for containing a solution containing metal ions separated by the insulating film, and two liquid reservoirs respectively. And two electrodes installed in the space, a voltage source for applying a voltage between the two electrodes, an ammeter capable of measuring the current due to metal ions passing through the small hole, and a third electrode installed near the small hole. , A fourth electrode, and the third and fourth electrodes
It is equipped with a device that can measure the conductivity between the electrodes.

In the apparatus for recognizing a fine array of DNA according to the constitution (C) of the present invention, a piezoelectric thin film having small holes, two liquid reservoirs containing a solution containing metal ions separated by the piezoelectric thin film, and the two liquid reservoirs Two electrodes installed on each, a voltage source for applying a voltage between the two electrodes, an ammeter capable of measuring the current due to metal ions passing through the small holes, a pair of piezoelectrics installed on both sides of the piezoelectric thin film A voltage source for applying a voltage is provided between the film control electrode and the piezoelectric film control electrode.

The above fine array recognition device can be used for molecular counting, molecular sieving, and molecular sorter.

[0020]

In the present invention, small holes are formed in the insulating film by using the semiconductor microfabrication technique to play the role of protein channel in the lipid bilayer. Compared to protein channels, they are safer in production and have better controllability. In addition, it is highly stable against changes over time and the pH of the solution. For example, the silicon nitride film does not deteriorate with time, has resistance to an acid or alkaline solution, and a buffer solution to be used is not selected. Further, when processed into a membrane, a film having high flatness and high strength is formed. This is due to the moderate internal stress of the silicon nitride film.

To form the small holes, etching with a focused ion beam apparatus or high resolution electron beam resist, for example, calixarene type electron beam resist is used.

The focused ion beam device is designed to measure metal ions emitted from an ion source of solid metal such as gallium by several nm.
It can be focused up to. When the insulating film or the semiconductor film is irradiated with the focused ion beam, it can be etched to form minute holes. The diameter of the hole is 10n
The diameter of the hole can be changed by adjusting the ion beam diameter or ion dose (Journal of Vacuum Science and Technology B, Vol. 15, pp. 2373 to 2).
378, 1997). By using this focused ion beam apparatus, the small hole according to the present invention can be easily produced. Furthermore, in a tungsten carbonyl gas atmosphere,
When gallium ions are irradiated, tungsten is deposited at the position where the beam is irradiated. From this, it is possible to form a minute electrode at a desired position.

A second method of forming small holes is to use a calixarene-based high resolution electron beam resist. The high resolution electron beam resist of the calixarene system has a high resolution.
(Applied Physics Letter, Vol. 68, Vol. 1
297 to 1299, 1996), 13 nm with chloromethyl calixarene (Applied Physics Letters, Vol. 77, pp. 301 to 303).
Page, 2000).

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Next, embodiments of the present invention will be described in detail with reference to the drawings. The fine array recognition apparatus of the present invention is shown in FIG.

Example 1 FIG. 1A shows the appearance of Example 1 of the present invention. The insulating film 11 is arranged on the substrate 10 provided with the flow path, and the insulating film 11 is covered with the glass cover 12
Is covered. The glass cover 12 is provided with a buffer inlet 13, a buffer outlet 15, and an input / output port for a sample inlet 14. Wiring connected to the electrodes extends from the insulating film 11 and is connected to the voltage source 16 and the ammeter 17.

FIG. 1B shows the internal structure of the first embodiment of the present invention. (1) A substrate 10 provided with a flow path 20, and (2)
Insulating film 11 formed with through holes 24 and 26 for communicating from anodes 21 and 23, cathode 22, buffer inlet 13 and buffer outlet 15 to channel 20, and small hole 25 communicating to sample inlet 14 and channel 20 ( 3) It is composed of a buffer inlet 13, a buffer outlet 15, and a glass cover 12 on which a sample inlet 14 is formed.

The sample solution and the buffer solution contain metal ions and are made conductive. The sample solution contacts the cathode 22 and has the same potential as the cathode, while the buffer solution contacts the anodes 21 and 23 and has the same potential as the anode. The potential difference between the sample solution and the buffer solution depends on the voltage source 1.
Controlled by 6. When metal ions or charged molecules pass through the small holes, a current flows through the circuit, and the current is detected by the ammeter 17.

FIG. 2A is a view of the vicinity of the small hole 25 on the insulating film 11 as seen from directly above, showing the electrode arrangement in the case of this embodiment. The cathode 22 is arranged so as to surround the small hole.

FIGS. 3 and 4 explain the operating principle of the first embodiment of the present invention. 3A and 4
A is a cross-sectional view near the small hole 25 penetrating the insulating film 11. Although the anode 21 and the cathode 22 are on the insulating film in FIG. 1,
In this figure, it is schematically shown in the solution.

In FIG. 3A, the solution 41 containing the sample and the buffer solution 42 are separated with the insulating film 11 as a boundary. Each solution contains positively or negatively charged metal ions. When a voltage is applied between the cathode 22 and the anode 21 using the voltage source 16, a solution 41 containing a sample having the same potential as the cathode 22 and a buffer solution 42 having the same potential as the anode 21.
A potential difference V is generated at. When a potential difference occurs at both ends of the small hole,
The negatively charged metal ions pass through the small holes and move along the potential gradient from the cathode side to the anode side. On the other hand, the positively charged metal ions pass through the small holes and move along the potential gradient from the anode side to the cathode side. As a result, the metal ions carry an electric charge, and a current is generated in the external circuit. The magnitude of the electric current is proportional to the number of metal ions passing through the small hole per unit time. The number of metal ions per unit time is proportional to the cross-sectional area S of the small holes and the potential difference V, and inversely proportional to the length L of the small holes.

FIG. 3A further shows the case where the molecule 44 is added to the solution 41 as a sample. Molecule 44 is small hole 2
It is assumed that it is smaller than the opening width of 5. A schematic current observed when a potential difference V is applied between the sample-containing solution 41 and the buffer solution 42 is shown in FIG. 3B. When the molecule A passes through the small hole, a part of the small hole is blocked, so that the apparent cross-sectional area S becomes small. Therefore, the number of metal ions passing through the small holes changes, and the current is modulated. The magnitude of the modulation of the current is proportional to the size of the molecule passing through the pore. By counting the number of times the current is modulated, the concentration of the sample molecule contained in the solution can be known, and the size of the molecule can be known from the magnitude of the modulation. The example in FIG. 3B shows that three sample molecules have passed through the small hole. It can be seen that this method can realize molecule counting and size measurement.

FIG. 4A shows the case where the single-stranded DNA 45 is added to the solution 41 as a sample. Single-stranded DNA consists of bases of different sizes: adenine (a), thymine (t),
It is a chain polymer in which guanine (g) and cytosine (c) are bound to the sugar-phosphate backbone. When the single-stranded DNA enters the small hole, the electric current is modulated, and the magnitude of the electric current varies depending on each base. Therefore, the base sequence can be determined by observing the modulation of the current. FIG. 4B schematically shows the observed current. In this example, the base sequence of DNA can be determined as ggtatgccaa ttattcaacg ttac 24.

The change in observed current can be increased by modifying any of the bases constituting DNA. For example, a nucleotide obtained by adding a fluorescent label to the guanine (g) is inserted at the position of guanine. When guanine to which the above-mentioned label is added passes through the small hole, the observed current increases, and the position of guanine in DNA can be determined more accurately. Besides guanine with a fluorescent label,
More accurate recognition can be achieved by using a base modified from adenine (a), thymine (t), and cytosine (c) with a label.

FIG. 6 shows a manufacturing process of the fine array recognition apparatus according to the first embodiment of the present invention. Using a silicon substrate as a substrate, a fine array recognition device was actually manufactured by using a semiconductor fine processing technology.

(FIG. 6A) The substrate used is a silicon substrate,
Silicon nitride film 102 on the surface, and further silicon oxide film 1
01, a silicon nitride film 104 is formed on the back surface by vapor phase epitaxy. The film thickness of the silicon nitride films 102 and 104 is about 10 nm to 100 nm, and the film thickness of the silicon oxide film is about 300 nm. The silicon nitride film 104 on the back surface is made of 25% tetramethylammonium halide (TMAH).
Etching is performed by using to form trenches 105, 106 and 107 until the silicon nitride film 102 is exposed. TM
Potassium hydroxide (KOH) may be used instead of AH. The areas between the trenches 105 and 106 and 107 and 106 likewise etch the silicon substrate. (FIG. 6B) Silicon nitride film membranes 114 and 11 in which a part of the silicon oxide film 101 on the surface is opened and openings 111, 112 and 113 are provided, and the silicon nitride film is exposed.
5, 116 are formed. (FIG. 6C) Gold electrodes 121 to 126 using vacuum deposition
To form. The film thickness of the gold electrode is about 5 nm to 100 nm. (FIG. 6D) A part of the silicon nitride film membranes 114, 115 and 116 is etched by a focused ion beam device to form one or more through holes 131, 133 and small holes 132. Instead of using the focused ion beam apparatus, electron beam exposure may be performed using calixarene or chloromethylated calixarene to form an etching mask, and a through hole may be formed by reactive ion etching. The through holes 131 and 133 may be patterned by using a photoresist and then etched by reactive ion etching or the like. By irradiating the small holes 132 with an ion beam in a focused ion beam device or an electron beam in a scanning electron microscope, contamination can be deposited and the size of the holes can be narrowed. Alternatively, the metal may be deposited by vacuum vapor deposition or electroplating. When the electroplating method is used, the number of metal ions passing through the small holes can be measured while the holes can be narrowed with good control. (FIG. 6E) A glass cover provided with a doorway is bonded to the upper surface, and a glass substrate is bonded to the back surface. Adhesion is performed by anodic bonding. The adhesion may be performed using a polymer such as PMMA.

A fine array recognition device can be manufactured through the above steps. FIG. 9A is an electron micrograph of the small holes of the manufactured fine array recognition device.

(Second Embodiment) A second embodiment of the present invention will be described. The external appearance and internal structure of Example 2 of the present invention are the same as those of FIGS. 1A and 1B except that the shape of the cathode 22 on the insulating film 11 and the two electrodes 31 and 32 are different.

FIG. 2B is a view of the vicinity of the small hole 25 on the insulating film 11 as seen from directly above, showing the electrode arrangement of this embodiment. Separately from the cathode 22, third and fourth electrodes 31 and 32 are arranged. The electrodes 31 and 32 are arranged close to each other with a small hole in between.

The third and fourth electrodes 31 and 32 allow the conductivity of the molecule to be measured as it passes through the small hole 25. The four types of bases in DNA are
Since the shapes of electron clouds are different, the conductivity is different. Therefore, the base sequence can be known from the conductivity between the third and fourth electrodes 31 and 32. Together with the current between the anode 22 and the cathode 21, it serves to complement the base sequence determination.

FIG. 7 shows a manufacturing process of a fine array recognition apparatus according to the second embodiment of the present invention. A silicon array substrate is used as a substrate, and a fine array recognition device is actually manufactured by using a semiconductor fine processing technique.

(FIG. 7A) The substrate used is a silicon substrate,
Silicon nitride film 102 on the surface, and further silicon oxide film 1
01, a silicon nitride film 104 is formed on the back surface by vapor phase epitaxy. The film thickness of the silicon nitride films 102 and 104 is about 10 nm to 100 nm, and the film thickness of the silicon oxide film is about 300 nm. The silicon nitride film 104 on the back surface is made of 25% tetramethylammonium halide (TMAH).
Etching is performed by using to form trenches 105, 106 and 107 until the silicon nitride film 102 is exposed. TM
Potassium hydroxide (KOH) may be used instead of AH. The areas between the trenches 105 and 106 and 107 and 106 likewise etch the silicon substrate. (FIG. 7B) A part of the silicon oxide film 101 on the surface is opened, openings 111, 112, 113 are provided, and silicon nitride film membranes 114, 115, 116 are formed. (FIG. 7C) Gold electrodes 121-126 using vacuum deposition
To form. The film thickness of the gold electrode is about 5 nm to 100 nm. Further, the electrode 127 is formed by using a focused ion beam device. (FIG. 7D) A part of the silicon nitride film membranes 114, 115 and 116 is etched by a focused ion beam device to form one or more through holes 131, 133 and small holes 132. Instead of using the focused ion beam device, electron beam exposure may be performed using calixarene or chloromethylated calixarene to form an etching mask, and through holes and small holes may be formed by reactive ion etching. When making the small hole 132, the electrode 127
The electrodes 134 and 135 are produced by cutting. The through holes 131 and 133 may be patterned by using a photoresist and then etched by reactive ion etching or the like. (FIG. 7E) A glass cover provided with a doorway is bonded to the upper surface, and a glass substrate is bonded to the back surface. Adhesion is performed by anodic bonding. The adhesion may be performed using a polymer such as PMMA. A fine array recognition device can be manufactured through the above steps. FIG. 9B is an electron micrograph of small holes of the fine array recognition device manufactured in Example 2.

(Third Embodiment) A third embodiment of the present invention will be described. The external appearance and internal structure of Example 2 of the present invention are the same as those of FIGS. 1A and 1B except that the shape of the cathode 22 on the insulating film 11 and the two electrodes 31 and 32 are different. Also,
The insulating film 11 is a piezoelectric thin film having a piezoelectric effect.

FIG. 2C is a view of the vicinity of the small hole 25 on the insulating film 11 as seen from directly above, showing the electrode arrangement of this embodiment. Separately from the cathode 22, electrodes 33 and 34 for controlling the piezoelectric thin film
Are placed. The electrodes 33 and 34 are arranged on the front surface and the back surface of the piezoelectric thin film 35, respectively.

FIG. 5 explains the operation principle of the third embodiment of the present invention. FIG. 5A is a sectional view near the small hole 25 penetrating the piezoelectric thin film 35. Although the anode 21 and the cathode 22 are on the insulating film in FIG. 1, they are schematically shown in the solution in this figure.

In FIG. 5A, the piezoelectric thin film 35 is divided into a solution 41 containing a sample and a buffer solution 42. Each solution contains positively or negatively charged metal ions. When a voltage is applied between the cathode 22 and the anode 21 using the voltage source 16, a solution 41 containing a sample having the same potential as the cathode 22 and a buffer solution 42 having the same potential as the anode 21.
A potential difference V is generated at. When a potential difference occurs at both ends of the small hole,
The negatively charged metal ions pass through the small holes and move along the potential gradient from the cathode side to the anode side. On the other hand, the positively charged metal ions pass through the small holes and move along the potential gradient from the anode side to the cathode side. As a result, the metal ions carry an electric charge, and a current is generated in the external circuit. The magnitude of the electric current is proportional to the number of metal ions passing through the small hole per unit time. The number of metal ions per unit time is proportional to the cross-sectional area S of the small holes and the potential difference V, and inversely proportional to the length L of the small holes.

Piezoelectric thin film control electrode 3 on the piezoelectric thin film 35
When a voltage is applied between 3 and 34, the size of the small holes in the piezoelectric thin film changes by a displacement amount 27 due to the inverse piezoelectric effect in which the piezoelectric thin film is compressed or expanded. Molecules or D in small holes
When NA passes, the current changes as described above,
A negative feedback voltage is applied between the piezoelectric thin film control electrodes so that the current is constant, that is, the number of passing metal ions is constant. As a result, there is no risk that large molecules will block the small pores, and the flow of molecules or DNA passing through the small pores will approach a certain level. Since the negative feedback voltage is proportional to the size of the base of the molecule or DNA, it is possible to know the presence / absence or size of the passing molecule and the type of the base of DNA by reading the negative feedback voltage.

FIG. 8 shows a manufacturing process of a fine array recognition apparatus according to the second embodiment of the present invention. A silicon array substrate is used as a substrate, and a fine array recognition device is actually manufactured by using a semiconductor fine processing technique.

(FIG. 8A) The substrate used is a silicon substrate,
Silicon nitride film 102 on the surface, and further silicon oxide film 1
01, a silicon nitride film 104 is formed on the back surface by vapor phase epitaxy. The film thickness of the silicon nitride films 102 and 104 is about 10 nm to 100 nm, and the film thickness of the silicon oxide film is about 300 nm. The silicon nitride film 104 on the back surface is made of 25% tetramethylammonium halide (TMAH).
Etching is performed by using to form trenches 105, 106 and 107 until the silicon nitride film 102 is exposed. TM
Potassium hydroxide (KOH) may be used instead of AH. The areas between the trenches 105 and 106 and 107 and 106 likewise etch the silicon substrate. (FIG. 8B) A part of the silicon oxide film 101 on the surface is opened and openings 111, 112 and 113 are opened to form silicon nitride film membranes 114, 115 and 116.
Subsequently, lead titanate (PZT) 150, which is a kind of piezoelectric thin film, is grown by the sputtering method. The film may be formed by a sol-gel method other than the sputtering method. The lead titanate (PZT) may be barium titanate or a mixed compound thereof. (FIG. 8C) A part of the silicon nitride film is etched by reactive ion etching so that both surfaces of the piezoelectric thin film are exposed. Gold electrodes 121 to 12 using the vacuum deposition method
6, 151 and 152 are formed. The film thickness of the gold electrode is about 5 nm to 100 nm. (FIG. 8D) A part of the silicon nitride film membranes 114, 115 and 116 is etched by a focused ion beam device to form one or more through holes 131, 133 and small holes 132. Instead of using the focused ion beam apparatus, electron beam exposure may be performed using calixarene or chloromethylated calixarene to form an etching mask, and a through hole may be formed by reactive ion etching. The through holes 131 and 133 may be patterned by using a photoresist and then etched by reactive ion etching or the like. (FIG. 8E) A glass cover provided with a doorway is adhered to the upper surface, and a glass substrate is adhered to the back surface. Adhesion is performed by anodic bonding. The adhesion may be performed using a polymer such as PMMA. A fine array recognition device can be manufactured through the above steps.

[0049]

According to the fine array recognition apparatus of the present invention, D
The base sequence of NA can be determined inexpensively, efficiently, and quickly.

Since the fine array recognition apparatus according to the present invention uses semiconductor fine processing, it is highly safe in production, and since biomolecules are not used as a material, there is little change with time and stability depending on the type of solution. Is high.

[0051]

[Sequence list] <110> NEC Corporation <120> Device and method for analysis of individual molecules <130> 34103599 <160> 2 <210> 1 <211> 24 <212> DNA <400> 1 ggtatgccaa ttattcaacg ttac 24

[Brief description of drawings]

FIG. 1 is an external view (A) and an internal structure (B) of a fine array recognition device.

FIG. 2 shows an electrode arrangement around a small hole. (A) Example 1, (B)
Example 2 (C) Electrode arrangement around the small holes of the fine array recognition device described in Example 3

FIG. 3A is a principle of operation as a molecule counting in Example 1 and FIG. 3B is an observed current.

4 (A) Operating principle of DNA sequencing of Example 1 and (B) Observed current.

5 (A) Operating principle of molecule counting in Example 3 and (B) Observed current.

FIG. 6 is a manufacturing process of the fine array recognition apparatus in Example 1

FIG. 7: Manufacturing process of a fine array recognition apparatus in Example 2

FIG. 8 is a manufacturing process of a fine array recognition apparatus of Example 3

FIG. 9 is a small hole portion of the fine array recognition device of (A) Example 1 and (B) Example 2;

[Explanation of symbols]

10. ． ． Substrate provided with flow channel 11. ． ． Insulating film 12. ． ． Glass cover 13. ． ． Buffer inlet 14. ． ． Sample inlet 15. ． ． Buffer outlet 16. ． ． Voltage source 17. ． ． Ammeter 20. ． ． Flow paths 21, 23. ． ． Anode 22. ． ． Cathode 24, 26. ． ． Through hole 25. ． ． Small hole 31. ． ． Third electrode 32. ． ． Fourth electrodes 33, 34. ． ． Piezoelectric film control electrode 35. ． ． Piezoelectric thin film 41. ． ． Sample solution 42. ． ． Buffer solution 43. ． ． Metal ion 44. ． ． Sample molecule 45. ． ． DNA 46, 47. ． ． Protective film 48. ． ． Voltage source 101. ． ． Silicon oxide films 102, 104. ． ． Silicon nitride film 103. ． ． Silicon substrates 105, 106, 107. ． ． Etching grooves 111, 112, 113. ． ． Openings 114, 115, 116. ． ． Silicon nitride film membranes 121, 122, 123, 124, 125, 12
6. ． ． Gold electrode 127. ． ． Tungsten electrodes 131, 132, 133. ． ． Etching holes 134, 135. ． ． Tungsten electrodes 141, 142. ． ． Glass cover 150. ． ． Lead titanate thin films 151, 152, 153, 154. ． ． Gold electrode

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/50 G01N 27/46 336M // C12N 15/09 ZNA 336B 336Z 336G C12N 15/00 ZNAA (72) Inventor Kazuhiro Iida 5-7-1, Shiba 5-chome, Minato-ku, Tokyo Inside NEC Corporation (72) Inventor Toru Sano 5-7-1, Shiba 5-chome, Minato-ku, Tokyo (72) Inventor Masakazu Baba 5-7-1 Shiba 5-chome, Minato-ku, Tokyo F-Term inside NEC Corporation (reference) 2G045 AA35 DA13 GC20 JA07 2G060 AA06 AC02 AC10 AD06 AE17 AF01 AF06 AF08 AG11 FA01 FB02 HA01 HC07 HC18 HE05 JA06 KA06 4B024 AA11 CA01 CA09 HA13 HA19 4B029 AA07 BB20 FA10 FA12 HA09 4B063 QA01 QA13 QA18 QQ42 QR50 QR56 QS22 QX02 QX04

Claims (15)

[Claims] 1. A device capable of determining the base sequence of DNA at high speed, comprising a plurality of liquid reservoirs separated through an electrically insulating film having small pores (channels) through which ions pass, and their respective liquid reservoirs. A fine array recognition apparatus comprising a plurality of electrodes for energizing a solution in a liquid reservoir, applying a voltage between any of the electrodes, and measuring a current generated by ions passing through a small hole. 2. The insulating film is a silicon nitride film, a silicon oxide film, or a silicon oxynitride film, or
The fine array recognition device according to claim 1, wherein the fine array recognition device is a silicon nitride film, a silicon oxide film, or a silicon oxynitride film exposed by etching a silicon substrate. 3. The measured current information is used to perform sequence structure determination, counting, sorting (sieving), sorting (classification) and retrieval of DNAs and molecules passing through the small holes. 2. The fine array recognition device according to any one of 2. 4. The fine array recognition device according to claim 1, wherein, in addition to a plurality of electrodes for energizing the solution, electrodes facing each other with a small hole in between are installed. . 5. The insulating film is a piezoelectric thin film, and electrodes are formed on both surfaces of the piezoelectric thin film.
The fine array recognition device according to any one of 2 and 3. 6. The small holes are formed by a focused ion beam (FIB) technique.
5. The method for manufacturing the fine array recognition device according to any one of 5 above. 7. The small hole is formed by using a high-resolution electron beam resist such as calixarene, chloromethylated calixarene, or polystyrene. A method for manufacturing a fine array recognition device according to claim 1. 8. The vacuum deposition method, the chemical vapor deposition method, or the electroplating method is used to reduce the size of the small holes after forming the small holes.
The method for manufacturing a fine array recognition device according to any one of 2, 3, 4, and 5, or the method for manufacturing a fine array recognition device according to any one of claims 6 and 7. 9. The size of a small hole is determined by measuring and controlling an ionic current between two solvents separated by an insulating film having a small hole when the electroplating method is used. 8. The method for manufacturing the fine array recognition device according to item 8. 10. A method or a method for manufacturing a fine array recognition apparatus according to claim 1, wherein a focused ion beam (FIB) technique is used in forming the electrodes. Item 10. A method for manufacturing a fine array recognition device according to any one of items 6, 7, 8 and 9. 11. The microarray or the DNA base passing through the small hole is recognized as one kind at a specific time in a specific small hole, according to claim 1, 2, 3, 4, 5. A fine array recognition method in the fine array recognition apparatus according to any one of claims. 12. The fine array recognizing method in a fine array recognizing apparatus according to claim 4, wherein the fine array is recognized by measuring conductivity between electrodes facing each other across the small hole. 13. A signal in which, when the insulating film is a piezoelectric thin film, the size of the small holes of the piezoelectric thin film changes due to an inverse piezoelectric effect when a voltage is applied between a pair of electrodes formed on both surfaces of the piezoelectric thin film. The fine array recognition method of the fine array apparatus according to claim 5, wherein is used as information for fine array recognition. 14. A negative feedback voltage is applied between the electrodes formed on both sides of the piezoelectric thin film to change the diameter of the small hole to keep the flow of ions passing through the small hole constant and to reduce the negative feedback voltage. By reading, the presence / absence of molecules passing through and the size, D
2. A type of NA base is recognized.
The fine array recognition method of the fine array recognition apparatus according to item 3. 15. When a specific base among DNA-constituting bases is modified to change the molecular size of the base, a change in ionic current at the moment when the modified base passes through a small hole is emphasized, and the base is modified. 6. The fine array recognition method in the fine array recognition apparatus according to claim 1, wherein the detection of [3] is facilitated.