JP4453331B2 - Apparatus and method for detecting electrical characteristics - Google Patents

Description

The present invention relates to an electrical characteristic sensing device and method for detecting an electrical characteristic of a nucleotide chain.

  In recent years, the analysis speed of a DNA sequencer, which is an apparatus for automatically reading a DNA base sequence, has been increased (for example, see Patent Document 1).

  However, at present, the analysis speed is still very slow, and if a single genome sequencer is used to analyze a human genome having about 3 billion bases, it still consumes several years or more.

JP-A-8-278281

It is an object of the present invention to provide an electrical property detection apparatus and method that can detect electrical properties of nucleotide chains at high speed.

An electrical property detection apparatus according to the present invention comprises a storage region for storing a nucleotide chain to be detected, a micro region formed in the storage region and wider than the stretched nucleotide chain, and an electrical property of the micro region. Detection means having a sensor for detecting a change in the physical characteristics, and an electric field applying means for generating electric lines of force that pass through the microregion, wherein the nucleotide chain passes through the microregion. In the meantime, a change in the electrical characteristics of the minute region is detected for each base.

The electrical property detection method according to the present invention stores a nucleotide chain to be detected, generates electric lines of force, thereby moving the nucleotide chain to a minute region wider than the diameter of the nucleotide chain, and moving the minute region to the nucleotide chain. While the signal is passing, the change in the electrical characteristics of the minute region is detected for each base.

In the above-described electrical property detection apparatus and method according to the present invention, a nucleotide chain to be detected is electrophoresed and extended. At the same time, the stretched nucleotide chain is moved by electrophoresis from one end side to a minute space having the same size as the diameter of the stretched nucleotide chain. A sensor for detecting electrical characteristics is provided in this minute space. Then, while the nucleotide chain passes through the minute space by electrophoresis, an electrical change in the minute space is detected, and the nucleotide sequence of the nucleotide chain is detected based on the detection result.

With the electrical property detection apparatus and method according to the present invention as described above, electrical properties can be detected at high speed.

As a best mode for carrying out the present invention, a DNA sequencer to which the present invention is applied will be described below. A DNA sequencer is a device that detects the base sequence of nucleotide chains such as ssDNA and oligonucleotide chains. Hereinafter, the nucleotide chain that is the detection target of the base sequence is simply referred to as target DNA.

  FIG. 1 shows a schematic diagram of a DNA sequencer 10 to which the present invention is applied. In the DNA sequencer 10, a solution holding part 12 that forms a hole-like space for holding or storing a buffer solution is formed on the surface of a base 11 made of an insulating member such as glass. A solution 13 such as water or a DNA buffer is held in the internal space or the internal region of the solution holding unit 12. In the solution 13, the target DNA 1 is dropped. The target DNA 1 is floating in the solution in the form of a random coil except during the detection operation of the DNA sequencer 10.

  The internal space of the solution holding unit 12 is open, for example, in a rectangular shape with respect to the surface of the substrate 11. A length t of one side of a predetermined direction of the opening (hereinafter, this direction is referred to as an X direction) is about 50 μm, and a length h in the depth direction is about 5 μm. In addition, the length t of the side in the X direction of the solution storage unit 12 is sufficiently longer than the length when the target DNA 1 is expanded.

  A cylindrical portion 15, which is a minute cylindrical member, is provided on one side surface 14 (hereinafter, referred to as a first side surface 14) in a direction orthogonal to the X direction of the solution storage portion 12. As shown in FIG. 2, the cylindrical portion 15 has one end in the direction of the central axis of the cylinder connected to the first side surface 14, and the central axis of the cylinder extends from the side surface 14 in the vertical direction (X direction). It is provided to stretch. For this reason, in the cylindrical portion 15, the opening 16 on the other end side that is not connected to the first side surface 14 and the cylindrical internal space 17 are positioned in the internal space of the solution storage portion 12. That is, the opening 16 and the internal space 17 are immersed in the solution 13.

As shown in FIG. 2, the cylindrical portion 15 is provided with a pair of sensors 21 and 22 made of, for example, a metal material on the side wall portion in the vicinity of the opening portion 16. The pair of sensors 21 and 22 are provided so as to face each other with the central axis of the cylindrical portion 15 interposed therebetween. The sensors 21 and 22 are connected to the voltage detection unit 23 via electric wirings 21a and 22a. The voltage detection unit 23 detects a voltage change (or capacitance change) between the sensors 21 and 22 and detects the base sequence of the target DNA 1 based on the detection result.

  Here, the diameter of the opening 16 of the cylindrical portion 15 is such that the stretched target DNA 1 can be inserted into the internal space 17 from the outside through the tube opening 16 and the stretched target DNA 1 passes. The size is such that the sensors 21 and 22 can detect a change in voltage (or capacitance) due to a change in base sequence. Details of the diameter of the opening 16 and the size of the sensor 21 will be described later. Further, the length (depth) T of the internal space 17 of the cylindrical portion 15 is such a length that at least a part of the entire length of the extended target DNA 1 can be accommodated in the internal space 17.

  The solution storage unit 12 is provided with an electric field applying electrode 25 on a first side surface 14 that is a side surface orthogonal to the X direction, and is orthogonal to the X direction at a position facing the first side surface 14 in the X direction. An electric field application electrode 26 is provided on the other side surface 24 (hereinafter referred to as the second side surface 24).

  The electric field application electrodes 25 and 26 are provided at positions facing the X direction in the electric field application electrode 25 and the electric field application electrode 26. The electric field application electrode 25 is electrically connected to one polarity electrode (for example, a positive electrode) of the power source 27, and the electric field application electrode 26 is connected to the other polarity electrode (for example, a negative electrode) of the power source 27. . The power source 27 generates AC power or pulse power having a predetermined frequency and power, and is applied between the electric field applying electrodes 25 and 26. For this reason, the electric field applying electrodes 25 and 26 apply an electric field in the X direction into the solution 13. Further, as shown in FIG. 3, the electric field applying electrodes 25 and 26 are provided so that part of the generated lines of electric force pass through the opening 16 of the cylindrical portion 15 and also through the internal space 17. . In order to configure the electric lines of force in this way, the electric field applying electrodes 25 and 26 may be provided on the extended straight line of the central axis of the cylindrical portion 15. Although details will be described later, since the target DNA 1 needs to be moved from the second side surface 24 side to the first side surface 14 side by electrophoresis, as the second side surface 24 approaches the first side surface 14 side, the target DNA 1 needs to be moved. The width (length in the direction perpendicular to the X direction) of the electric field application electrode 24 provided on the second side surface 24 is widened so that the width of the electric lines of force (length in the direction perpendicular to the X direction) is reduced. It is desirable to narrow the width (the length in the direction perpendicular to the X direction) of the electric field applying electrode 14 provided on the first side face 14.

The operation of detecting the base sequence of the target DNA 1 by the DNA sequencer 10 as described above will be described.

  First, as shown in FIG. 4, the target DNA 1 is dropped together with the solution 13 into the internal space in the solution holding unit 12. The dropped target DNA 1 floats in the solution 13 in a random coil shape.

  Subsequently, as shown in FIG. 5, the power supply 27 is turned on, AC power or pulse power is applied between the electric field application electrodes 25 and 26, and electric lines of force E perpendicular to at least the central axis of the cylindrical portion 15. An AC electric field or a pulse electric field (for example, about 1 MHz, about 1 V / μm) is applied to the solution 13. When such an alternating electric field or a pulse electric field is applied, the target DNA 1 extends along the direction of the electric force lines and moves along with the direction of the electric force lines. As a result, the target DNA 1 extends in the X direction of the solution holding unit 12 and moves in the direction from the second side surface 24 toward the first side surface 14 as indicated by the white arrow in FIG.

Subsequently, as shown in FIG. 6, the extended end portion of the target DNA 1 is inserted into the inside through the opening portion 16 of the cylindrical portion 15, and then the target DNA 1 is sequentially inserted into the internal space 17. At this time, since the target DNA 1 passes between the sensors 21 and 22, the voltage (or capacitance) between the sensors 21 and 22 changes according to the base sequence. The voltage detector 23 detects a voltage change (or capacitance change) between the sensors 21 and 22 while the target DNA 1 is passing through the sensors 21 and 22 by electrophoresis, that is, during movement. Then, the voltage detection unit 23 detects the base sequence of the target DNA 1 based on the detected voltage change (or capacitance change).

As described above, in the DNA sequencer 10, the target DNA 1 is allowed to pass through the minute space by electrophoresis, and the base sequence of the target DNA 1 is directly detected by the sensor provided in the minute space. This makes it possible to analyze the base sequence of the target DNA 1 at a very high speed.

  Next, the size of the cylindrical portion 15 of the DNA sequencer 10 and the electrophoresis speed of the target DNA 1 are verified.

  FIG. 7 schematically shows the relationship between the sensors 21 and 22 and the extended target DNA 1.

First, in order to detect the base sequence of the target DNA 1 by the sensors 21 and 22, it is necessary to be able to detect a minute voltage (or capacitance) generated from each base of the target DNA 1. For this purpose, when the distance between the pair of sensors 21 and 22 is W and the diameter of the stretched target DNA 1 is r, the relationship between W and r is detected when “W / r >> 1”. It cannot be detected unless “W / r≈1”. That is, since the diameter r of the extended nucleotide chain is about 2 nm, the distance W between the pair of sensors 21 and 22 is on the order of nanometers or tens of nanometers (for example, about 2 nm to 10 nm). It is desirable. However, W must be r or more (for example, 2 nm or more) because the extended nucleotide chain must be able to pass between the sensors.

  In order to set the distance W between the sensors 21 and 22 to the nanometer order in this way, the cylindrical portion 15 may be formed by using a so-called nano technology such as a silicon nanowire or a carbon nanotube (for example, Y Huang, X. Duan, Q. Wei, CM Lieber, Science 291, 26, January, 630, 2001).

  For example, in order to form the cylindrical portion 15 using the silicon nanowire technology, as shown in FIG. 8, a gold fine particle 31 of about 2 mm is attached to the first side face 14, and silane gas is introduced in the X direction as shown in FIG. It is possible to form the cylindrical portion 15 by flowing it through.

  And what is necessary is just to form the sensors 21 and 22 in the front-end | tip part of such a cylindrical part 15 using a semiconductor technique, for example.

Further, in order to detect the base sequence of the target DNA 1 by the sensors 21 and 22, the width between the pair of sensors 21 and 22 is L, and the length of one base constituting the extended target DNA 1 is d. In this case, the relationship between L and d is preferably “L / d≈1”. That is, since the length of one base of the nucleotide chain is about 0.35 nm, it is desirable that the sensor width L is on the order of this level. However, the sensor width that can be formed by the current semiconductor technology is on the order of several tens of nanometers, which is very difficult.

Therefore, the present inventor has decided to detect the voltage change (capacitance change) for each base while extending the extended target DNA 1 by an AC electric field or a pulse electric field and moving between the sensors at a predetermined speed. In other words, if the voltage (capacitance) can be sampled at least once within the period in which the extended target DNA 1 moves by one base, the sensor width L may be longer than the base length d. By detecting the voltage change amount, the base sequence of the target DNA 1 can be analyzed.

Specifically, the speed of the target DNA 1 by electrophoresis is V _e = about 1.4 × 10 ⁻⁴ (m / second) when the applied electric field is, for example, 500 (V / m). Therefore, the transfer time for one base (d / V _e ) is
d / V _e = 0.35 (nm) /1.4×10 ⁻⁴ (m / sec)
= About 2.5 μsec.

Even with the current analog / digital sampling technology, voltage sampling of several tens to several hundreds of times is possible within a period of about 2.5 μsec, and the base sequence of the target DNA 1 is detected by the DNA sequencer 10. It is understood that is possible.

It is a schematic diagram of a DNA sequencer to which the present invention is applied. It is a schematic diagram of a cylindrical part. It is a figure which shows the electric field applied by an electric field application electrode. It is a figure which shows the state by which target DNA was dripped in the solution. It is a figure which shows a state when an electric field is applied in a solution. It is a figure which shows the state which has detected the base sequence of target DNA. It is a figure which shows the relationship between a sensor and the extended target DNA. It is a figure which shows the state which attached the gold particle in order to produce | generate a silicon nanowire. It is a figure which shows the state which flowed silane gas in order to produce | generate a silicon nanowire.

  DESCRIPTION OF SYMBOLS 1 Target DNA, 10 DNA sequencer, 11 Base | substrate, 12 Solution holding | maintenance part, 13 Solution, 14 1st side surface, 15 Cylindrical part, 16 Opening part, 17 Internal space, 21, 22 Sensor, 23 Voltage detection, 24 2nd Side, 25, 26 Electric field application electrode, 27 Power supply

Claims (6)

A storage region for storing a nucleotide chain to be detected;
A microregion formed in the storage region and wider than the extended diameter of the nucleotide chain;
Detection means having a sensor for detecting a change in electrical characteristics of the micro area;
Electric field application means for generating electric lines of force that pass through the minute region,
Said detecting means, while the small region above nucleotide chains have passed, the electrical characteristic detecting apparatus characterized by detecting a change in electrical properties of the microscopic regions for each base. The electric field applying means applies an alternating electric field to the minute region,
2. The electrical property detection apparatus according to claim 1, wherein the nucleotide chain is subjected to electric field expansion and the minute region is moved by electrophoresis. 2. The electrical property detection device according to claim 1, wherein the electric field applying means applies an alternating electric field so as to concentrate the lines of electric force from the storage region to the minute region. 2. The minute region is a region through which the nucleotide chain can be passed, which is covered with a conductive material for the detection means to detect a change in the electrical characteristics. Electrical characteristic detection device. The electrical property detection device according to claim 4, wherein the minute region is a hollow, substantially cylindrical region. Store the nucleotide chain to be detected,
By generating electric lines of force, it moves to a small region wider than the diameter of the nucleotide chain,
While the micro-region is the nucleotide chain has passed, the electrical characteristic detecting method characterized by detecting a change in electrical properties of the microscopic regions for each base.

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