JP2004061426A - Base sequence detector and base sequence automatic analyzer - Google Patents

Abstract

An object is to improve in-plane uniformity in a cell.
The chip includes a plurality of electrodes to which a DNA probe is immobilized, a base sequence detection chip for defining a cell bottom surface, and a seal material for contacting the chip from the bottom surface of the seal material to the top surface of the seal material. A seal member 24 provided so as to surround the plurality of electrodes 21a by the inner periphery and defining a cell side surface; a chip cartridge upper lid 112 abutting on the seal material 24 upper surface to close the seal material 24 upper surface and define the cell upper surface; An inlet port 116a that opens on the upper surface and feeds a chemical solution or air into the cell 115, an outlet port 116b that opens on the upper surface of the cell and sends a chemical solution or air from inside the cell, and a chip cartridge upper lid 112 are provided. And a sample inlet 119 for injecting a sample into the cell, and the flow path of the chemical or air from the inlet port 116a to the outlet port 116b is narrow. It becomes formed in a shape.
[Selection diagram] FIG.

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a base sequence detection device for detecting a base sequence, and a base sequence automatic analyzer for automatically controlling the base sequence detection device and automatically analyzing a measurement signal.
[0002]
[Prior art]
Conventionally, for example, there is an apparatus that performs only hybridization, an apparatus that performs only electrochemical measurement after adding an intercalating agent after the hybridization, or an apparatus that automatically performs steps from hybridization to washing with a buffer. Was.
[0003]
[Problems to be solved by the invention]
When the measurement is performed using the apparatus as described above, when each step is completed, the operator is required to manually transfer the sample to the apparatus for the next step, which is time-constrained. In addition, since an operator is involved in transfer between processes, there is a problem that data reproducibility between samples is poor.
[0004]
On the other hand, there is also a problem that the measurement result varies depending on the reaction conditions in the cell for performing the reaction. This is considered to be due to, for example, in-plane non-uniformity of the reaction in the cell in which the electrochemical reaction is performed.
[0005]
The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a base sequence detection device that improves in-plane uniformity in a cell.
[0006]
Another object of the present invention is to provide a base sequence automatic analyzer that can automatically perform from reaction to liquid sending and measurement.
[0007]
[Means for Solving the Problems]
According to one aspect of the present invention, there is provided a base sequence detection device for detecting a base sequence in a sample by an electrochemical reaction between a sample and a probe having a predetermined base sequence in a flow path, comprising: a substrate; A plurality of base sequence detection chips provided with a plurality of electrodes to which the probes are immobilized, and a base solution detection chip provided on the substrate so as to surround the plurality of electrodes, and a chemical solution or air is sealed on the substrate. A cell, a delivery port that opens on the upper surface of the cell and sends a chemical solution or air into the cell, a delivery port that opens on the upper surface of the cell, and sends a solution or air from inside the cell, and the cell A sample injection port provided to open on the upper surface and injecting a sample into the cell, wherein the flow of the chemical solution or air from the inlet port formed in the cell to the outlet port is provided. What is a road The width of the straight direction of the cell, the feed base sequence detecting device in which is smaller than the distance of the incoming port and the delivery port is provided.
[0008]
According to another aspect of the present invention, a base for detecting a base sequence in a sample by an electrochemical reaction between a sample and a probe having a predetermined base sequence in a cell defined by a cell top surface, a cell side surface, and a cell bottom surface An array detection device, comprising: a substrate; a plurality of electrodes formed on the substrate, to which the probes are fixed; a base sequence detection chip defining the cell bottom surface by the substrate surface; A seal member is provided so as to surround the plurality of electrodes by a seal material inner circumference from a seal material bottom surface to be contacted to a seal material upper surface having a predetermined height, and abuts on the seal material defining the cell side surface and the seal material upper surface. To seal the upper surface of the sealing material and open the upper surface of the chip cartridge upper lid that defines the upper surface of the cell, and a chemical solution or liquid in the cell. An inlet port for sending air, an opening on the upper surface of the cell of the chip cartridge upper cover, a sending port for sending a chemical solution or air from inside the cell, and a transfer port provided on the chip cartridge upper cover, and a sample in the cell. And a sample injection port to be injected, wherein the width of the cell in a direction perpendicular to the direction of the flow path of the drug solution or air from the inlet port formed in the cell to the outlet port, There is provided a base sequence detection device formed to be smaller than the distance between the input port and the output port.
[0009]
According to still another aspect of the present invention, there is provided a device for detecting a base sequence as described above, which is connected to the inlet port, and supplies a chemical solution or air into the cell via the inlet port. And a supply system including a first valve for controlling a flow rate of a chemical solution or air in the first pipe, and a chemical solution or air from the inside of the cell through the delivery port, communicating with the delivery port. And a second valve for controlling a flow rate of a chemical solution or air in the second piping, and a pump provided in the second piping and sucking up a chemical solution or air from inside the cell. A discharge system, a measurement system including a voltage application unit for applying a voltage between the plurality of electrodes and another counter electrode, a temperature control mechanism for controlling the temperature of the base sequence detection chip, and the liquid sending system. The first valve, the second valve, and the pump of the measurement system; The control unit controls the application unit and the temperature control mechanism, detects an electrochemical reaction signal from the plurality of electrodes, and stores the electrochemical reaction signal as measurement data, and provides a control condition parameter to the control mechanism. And a computer that controls the control mechanism and executes a base sequence analysis process based on the measurement data.
[0010]
Further, the present invention relating to an apparatus is also realized as an invention of a method realized by the apparatus.
[0011]
Further, the present invention according to an apparatus or a method is realized as a program for causing a computer to execute a procedure for controlling the apparatus, and a computer-readable recording medium on which the program is recorded.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0013]
FIG. 1 is a conceptual diagram showing an overall configuration of an automatic base sequence analysis apparatus according to one embodiment of the present invention. As shown in FIG. 1, the automatic base sequence analyzer 1 includes a chip cartridge 11 (base sequence detector), a measurement system 12 electrically connected to the chip cartridge 11, and a flow path provided in the chip cartridge 11. And a temperature control mechanism 14 for physically controlling the temperature of the chip cartridge 11 and a liquid feeding system 13 physically connected via an interface unit.
[0014]
The measurement system 12, the liquid feeding system 13, and the temperature control mechanism 14 are controlled by a control mechanism 15. The control mechanism 15 is electrically connected to a computer 16, and the control mechanism 15 is controlled by a program provided in the computer 16. In the present embodiment, the chip cartridge 11, the measurement system 12, the liquid supply system 13, and the temperature control mechanism 14 are referred to as a measurement unit 10.
[0015]
A printed circuit board 22 on which a base sequence detection chip 21 to which a DNA probe is immobilized is mounted is used for the chip cartridge 11.
[0016]
In the following embodiments, the base sequence of the DNA to be detected is referred to as a target base sequence. Then, the base sequence having complementarity with the target base sequence and selectively reacting with the target base sequence is referred to as a target complementary salt sequence. The DNA probe containing the target complementary base sequence is immobilized on the working electrode of the base sequence detection chip 21. The sample (sample solution) introduced into the cell of the base sequence detection chip 21 contains DNA to be tested. The base sequence of the DNA to be tested is called a sample base sequence.
[0017]
The base sequence detection device of this embodiment hybridizes the sample base sequence with the target complementary base sequence, buffers the presence or absence of the reaction, and monitors after the introduction of the intercalating agent, so that the target base sequence is contained in the sample. Is determined.
[0018]
2A and 2B are diagrams showing a detailed configuration of the chip cartridge 11, wherein FIG. 2A is a view from the top, FIG. 2B is a view from the AA direction, and FIG. 2C is a view from the BB direction. FIG. 4D is a partial perspective cross-sectional view, and FIG. 4D is a view of a support 111, which is one component of the chip cartridge 11, as viewed from the back.
[0019]
The chip cartridge main body 110 includes a support 111 for supporting the printed circuit board 22 from the lower side, and a chip cartridge upper lid 112 for sandwiching and supporting the printed circuit board 22 from the upper side together with the support 111.
[0020]
Two openings are provided on the side of the chip cartridge upper lid 112, and one of the openings is connected to the interface unit 113a, and the other is connected to the interface unit 113b. These interface units 113a and 113b function as an interface between the liquid feeding system 13 and the chip cartridge 11.
[0021]
Channels 114a and 114b are provided inside the interface units 113a and 113b, respectively. A chemical solution or air from the upstream side of the liquid sending system 13 is sent into the chip cartridge 11 via the flow path 114a. The sample, the chemical solution, and the air in the chip cartridge 11 are sent to the downstream side of the liquid sending system 13 via the flow path 114b.
[0022]
2A to 2C, the flow paths 114a and 114b are indicated by broken lines. These flow paths 114a and 114b communicate from the interface portions 113a and 113b to the inside of the chip cartridge upper lid 112, and further to a cell 115 indicated by a circular broken line in FIG. 2A. The cell 115 is an area provided for causing an electrochemical reaction between the base sequence detection chip 21 and various solutions introduced into the base sequence detection chip 21. When the four corners of the printed board 22 on which the base sequence detection chip 21 is mounted are fixed to the chip cartridge upper lid 112 of the chip cartridge 11 by the board fixing screws 25, the cell 115 is sealed with the base sequence detection chip 21. The material 24 is defined by a closed space region surrounded by the chip cartridge upper lid 112. In a state where the printed board 22 on which the base sequence detection chip 21 is mounted is fixed to the chip cartridge upper lid 112, the printed board 22 is held by the support 111 and the chip cartridge upper lid 112 with the sealant 24 interposed therebetween. Further, the chip cartridge upper lid 112 is fixed by the upper lid fixing screw 117. Thereby, the injection / discharge paths of various chemicals and air that are communicated from the flow path 114a to the flow path 114b via the cell 115 are determined. The base sequence detection chip 21 is sealed on a printed board 22 with a sealing resin 23.
[0023]
The chip cartridge upper lid 112 located on the upper surface of the cell 115 is provided with an inlet port 116a and an outlet port 116b. The inlet port 116a penetrates from the side surface to the bottom surface of the chip cartridge upper cover 112, and opens to the bottom surface of the chip cartridge upper cover 112 at the cell hole 115a. The delivery port 116b penetrates from the other side surface to the bottom surface of the chip cartridge upper cover 112, and opens to the bottom surface of the chip cartridge upper cover 112 at the cell hole 115b. By connecting the inlet port 116a to the channel 114a and the outlet port 116b to the channel 114b, the channel 114a communicates with the cell 115, and the channel 114b communicates with the cell 115.
[0024]
An electrical connector 22a is set on the surface of the printed board 22 at a position separated from the cell 115. The electrical connector 22a is electrically connected to the lead frame of the board main body of the printed board 22. The lead frame of the substrate body is electrically connected to various electrodes of the base sequence detection chip 21 by leads and the like. By connecting a terminal of the measurement system 12 to the electric connector 22a, an electric signal obtained by the base sequence detection chip 21 can be transmitted through a predetermined terminal provided at a predetermined position on the printed circuit board 22, and further to the electric connector. The signal can be output to the measurement system 12 via 22a.
[0025]
As shown in FIG. 2D, the support 111 has a U-shape, and a cutout 111a is provided in the center. The cutout 111a is smaller than the printed board 22 and larger than the base sequence detection chip 21. Thus, the temperature control mechanism 14 can be disposed in contact with the base sequence detection chip 21 without the support 111 while maintaining the support function of the printed board 22 by the support 111. Reference numeral 117a denotes a screw hole to which an upper cover fixing screw 117 is fixed.
[0026]
As the temperature control mechanism 14 for adjusting the temperature of the base sequence detection chip 21, for example, a Peltier element is used. Thereby, temperature control of ± 0.5 ° C. is possible. The reaction of DNA is generally performed in a temperature range relatively close to room temperature. Therefore, the temperature control using only the heater has poor stability. Further, since it is necessary to control the reaction of DNA by the temperature profile, a cooling mechanism is required separately. In that regard, the Peltier element is optimal because it can perform both heating and cooling by changing the direction of the current.
[0027]
FIG. 3 is a diagram showing the support 111 and the chip cartridge upper lid 112 before being fixed by the upper lid fixing screw 117. As shown in FIG. 3, four corners of a printed board 22 on which the base sequence detection chip 21 is mounted are fixed to the chip cartridge upper lid 112 with board fixing screws 25. The sealant 24 is integrated with the chip cartridge upper lid 112. Therefore, a cell 115 surrounded by the sealant 24 and the chip cartridge upper lid 112 is defined on the base sequence detection chip 21. Furthermore, the chip cartridge upper lid 112 is used by being fixed to the support 111 with the upper lid fixing screw 117. The board fixing screw 25 may be fixed from the back side of the printed board 22 or may be fixed from the front side. In this manner, by fixing the printed circuit board 22 to the chip cartridge upper lid 112, the adhesion between the base sequence detection chip 21, the sealant 24, and the chip cartridge upper lid 112 can be reliably maintained.
[0028]
FIG. 4 is a diagram showing a detailed configuration of the printed circuit board 22 on which the base sequence detection chip 21 is mounted. As shown in FIG. 4, a base sequence detection chip 21 is sealed on a printed board 22 by a sealing resin 23. On the base sequence detection chip 21, for example, an electrode 21a operating as a working electrode or a counter electrode and an electrode 21b operating as a reference electrode are provided. FIG. 4 schematically shows a case in which one electrode 21a is provided in each of four regions divided by the electrode 21b. However, one or more working electrodes and counter electrodes may be provided in each region. Of course. Further, the shape of the electrode 21b is not limited to that shown in FIG. Also, for reference, FIG. 4 shows a region where the cell 115 is defined by a broken line. As shown in FIG. 4, the cell 115 is defined such that the electrodes 21a and 21b are located in the area of the cell 115.
[0029]
An electrical connector 22 a is provided at an end of the printed circuit board 22. The electrodes 21a and 21b of the base sequence detection chip 21 and the electrical connector 22a are electrically connected by a lead frame provided on the surface of the printed circuit board 22 or the like. By connecting the signal interface of the measurement system 12 to the electric connector 22a, each electrode of the base sequence detection chip 21 and the measurement system 12 can be electrically connected.
[0030]
FIG. 5A is a cross-sectional view of the cell 115 shown in FIG. 2A and a chemical solution supply system leading to the cell 115 as viewed from the C-C direction. FIG. 5B is a top view of the vicinity of the cell 115.
[0031]
As shown in FIG. 5A, a closed space formed by tightly fixing the sealing material 24 such as a packing ring by the chip cartridge upper lid 112 and the base sequence detection chip 21 is the cell 115. The bottom surface of the cell 115 is determined by the surface of the base sequence detection chip 21. The side surface of the cell 115 is defined by the sealing material 24, and the upper surface of the cell 115 is defined by the lower surface of the chip cartridge upper lid 112. The bottom surface of the sealing material 24 is in contact with the base sequence detection chip 21 without any gap, and the top surface of the sealing material 24 is in contact with the bottom surface of the chip cartridge upper lid 112 without any gap. Thus, a completely closed space other than the cell holes 115a and 115b is defined, and the liquid tightness between the base sequence detection chip 21 and the lid 120 is maintained.
[0032]
The cross-sectional shape of the cell 115 has a circular shape as shown in FIG. An input port 116a and an output port 116b having a circular cross section are provided on the outer edge of the circular cell 115 at substantially symmetric positions with respect to the center of the circle of the cell 115. The outer circumferences of the input port 116a and the output port 116b are almost in contact with the outer circumference of the cell 115. The central axes of the circular cross sections of the inlet port 116a and the outlet port 116b are located on a straight line 115c passing through the center of the circular bottom surface of the cell 115.
[0033]
The inlet port 116a and the outlet port 116b each extend upward from the upper surface of the cell 115 to a predetermined height in a direction substantially perpendicular to the cell bottom surface. The inlet port 116a and the outlet port 116b have their flow paths bent in a direction away from the center of the cell 115, and are connected to the flow paths 114a and 114b, respectively.
[0034]
As shown in FIG. 5B, the central axes of the inlet port 116a and the outlet port 116b, and the flow paths 114a and 114b are substantially located on a straight line 115c. The delivery port 116b extends to a predetermined height in a direction substantially perpendicular to the cell bottom surface, and is further bent at a substantially right angle in a direction away from the center of the cell 115, and branches into two paths at the bent position. One of the paths penetrates to the surface of the chip cartridge upper lid 112 and communicates with the sample inlet 119. As a result, the sample injected from the sample inlet 119 is introduced into the cell 115 through the delivery port 116b. The central axes of the sample inlet 119 and the delivery port 116b substantially coincide with each other, and the diameter of the sample inlet 119 is set larger than the diameter of the delivery port 116b. Further, the sample inlet 119 can be closed by the lid 120 provided near the sample inlet 119. Accordingly, when the chemical solution is circulated from the flow path 114a to the flow path 114b via the cell 115 without using the sample injection port 119, the chemical liquid can be prevented from flowing out from the sample injection port 119, and the chemical liquid can be prevented from flowing out. A route can be secured. Further, a sealing material 121 is provided on the lid 120, and by sealing the sample inlet 119, a slight leakage of the chemical solution can be prevented. Although not particularly shown in the example of FIG. 5A, a sealing material 121 having a depth that completely blocks the path from the delivery port 116b to the flow path 114b to the sample inlet 119 is left. If used, the retention of a chemical solution or air on the sample inlet 119 side can be reduced.
[0035]
With the above-described configuration, the liquid medicine and air can flow in the direction indicated by the arrow in FIG. 5A in the order of the flow path 114a, the inlet port 116a, the cell 115, the output port 116b, and the flow path 114b. The sample is injected from the sample inlet 119, and is introduced into the cell 115 through the delivery port 116b in the direction of the arrow. Therefore, the sample is injected from the sending side, and the flow of the supply of the chemical solution and the injection path of the sample are set in reverse. Thereby, in the cleaning step, the cleaning efficiency of the sample can be increased.
[0036]
FIG. 6A is a modified example of a portion surrounded by a broken line in FIG. 5A, and FIG. 6B is a view of the cell 115 in the modified example of FIG. As shown in FIG. 6A, the inlet port 116a has a counterbore hole 115d. In other words, the diameter of the inlet port 116a gradually increases toward the counterbore hole 115d, and the diameter of the port far from the opening of the inlet port 116a is smaller than the diameter of the counterbore hole 115d. When viewed from above, the positional relationship is as shown in FIG. The counterbore hole 115d has a diameter d of the inlet port 116a. ₁ Larger diameter d ₁₁ Having. The outer periphery of the inlet port 116a and the inner periphery of the sealing material 24 are arranged substantially in alignment. Therefore, a part of the outer periphery of the counterbore hole 115d is positioned so as to overlap the peripheral edge of the cell 115. The outer periphery of the counterbore hole 115d does not need to be circular, and as shown in FIG. 6B, an oval shape in which the hole width in the direction parallel to the straight line 115c is set smaller than the hole width in the direction perpendicular to the straight line 115c. May be.
[0037]
Although FIG. 6 shows a case where the counterbore hole 115d is provided in the sending port 116a, a similar counterbore hole may be provided in the sending port 116b.
[0038]
By providing the counterbore hole 115d at the opening of the cell 115, the inlet to the cell 115 has a funnel-like shape, so that it is easy to suck out a chemical solution or bubbles, and it is difficult for the solution or bubbles to remain in the cell 115. Has an effect.
[0039]
FIGS. 7A and 7B are diagrams showing a more detailed configuration of the positional relationship between components near the cell 115. FIG. 7A is a sectional view, and FIG. 7B is a top view. In FIG. 7A, d ₄ Is the height of the sealing material 24. In FIG. 7B, d ₀ Is the distance from the central axis O of the cell 115 to the outer periphery, d ₃ Is the outer diameter of the sealing material 24, d ₁ , D ₂ Are the port cross-sectional radii of the inlet port 116a and the outlet port 116b, respectively, and d ₁ <D ₃ , D ₂ <D ₃ It is. The central axes of the inlet port 116a and the outlet port 116b are ₁ And O ₂ And axis O and axis O ₁ Distance of d ₀₁ , Axis O and axis O ₂ Distance of d ₀₂ Then d ₀₁ + D ₁ = D ₀ , D ₀₂ + D ₂ = D ₀ Holds. Radius d of cell 115 ₀ Is set to about 3 mm to 30 mm, and the radius d of the input port 116a and the output port 116b is ₂ , D ₃ Are set to about 0.3 mm to 2 mm, respectively. Height d of sealing material 24 ₄ Is set to about 0.5 mm.
[0040]
As described above, by opening the inlet port 116a and the outlet port 116b at the peripheral edge of the upper surface of the cell 115 having a circular bottom, the chemical liquid is displaced when the inside of the cell 115 is replaced with air when the chemical liquid is supplied. 115, it is possible to prevent the chemical solution remaining. Also, when a chemical solution is introduced into the cell 115 filled with air, the cell 115 can be filled without remaining air. Further, since the sample inlet 119 is also provided so as to communicate with the delivery port 116b, it is possible to prevent air from remaining in the cell 115 during sample injection and to fill the cell 115 with the sample.
[0041]
Thereby, the in-plane uniformity of the current characteristics obtained in the cell 115 by the electrochemical reaction of the DNA probe in the cell 115 is improved, and the reliability of SNPs detection is improved.
[0042]
FIG. 7C shows a state in which the sealing material 24 is tightly fixed between the chip cartridge upper lid 112 and the base sequence detection chip 21 by the substrate fixing screw 25. It is pressed when the substrate fixing screw 25 is fixed in the direction indicated by the arrow in FIG. More specifically, after the sealant 24 is placed on the base sequence detection chip 21 and the chip cartridge upper lid 112 is fixed with the substrate fixing screw 25, the space between the upper surface of the sealant 24 and the chip cartridge upper lid 112 is changed. Contact is made without a gap, and the gap between the lower surface of the sealing material 24 and the surface of the base sequence detection chip 21 is made without a gap, and the cell 115 is defined.
[0043]
A receiving hole may be provided outside the cell hole portions 115a and 115b of the chip cartridge upper lid 112 for positioning the outer periphery of the inlet port 116a and the outlet port 116b of the chip cartridge upper lid 112 and the inner periphery of the sealant 24. . As a result, the sealant 24 fits into the receiving hole, and the sealant 24 is positioned with respect to the chip cartridge upper lid 112.
[0044]
FIG. 8 is a diagram showing a modification of each component near the cell 115 shown in FIG. FIG. 8A is a sectional view, and FIG. 8B is a top view. The same components as those in FIG. 7 are denoted by the same reference numerals, and detailed description is omitted. FIG. 8 differs from FIG. 7 in that the height d is ₄₁ Is used, and the chip cartridge upper cover 112 has substantially the same inner diameter and outer diameter as the seal material 24a, and the height d provided on the bottom surface of the chip cartridge upper cover 112. ₄₂ Is to provide the ring-shaped convex portion 112a. The ring-shaped convex portion 112a has the same central axis as the central axis O of the cell 115, and has an inner diameter d. ₀ , The outer diameter is d ₃ It is. The seal member 24a is printed in advance on the ring-shaped convex portion 112a by, for example, screen printing or the like, and is formed integrally with the seal member 24a. Thus, the cell 115 can be determined without positioning the sealing material and the chip cartridge upper lid as in the configuration shown in FIG. 7, and the assembly process of the cell 115 is simplified.
[0045]
As shown in FIG. 8C, the chip cartridge upper lid 112 having the sealing material 24a fixed in advance is fixed to the base sequence detection chip 21 by pressing the upper lid fixing screw 117 in the direction of the arrow using the upper lid fixing screw 117. The cell 115 can be easily defined. Also, height d ₄₁ And d ₄₂ Is the height d of the sealing material in FIG. ₄ For d ₄ = D ₄₁ + D ₄₂ The cell 115 having the same height as that in the case of FIG. 7 can be determined. Further, the sealing member 24a and the ring-shaped convex portion 112a may be integrated by a method other than screen printing. For example, the protrusions may be provided on one of the sealing member 24a or the ring-shaped convex portion 112a, and the other may be provided with a hole to be fitted with the protrusion to achieve the integration. In this case, there is an advantage that the sealing material 24a can be freely removed from the chip cartridge upper lid 112.
[0046]
FIGS. 9 to 11 show sectional views of modified examples of the shape of the cell 115. FIG.
[0047]
As shown in FIG. 9A, the cross section (bottom surface) of the cell 115 has a radius d. ₀ And has an outer diameter d surrounding the cell 115. ₃ Is disposed. The configuration shown in FIG. 9A is common to the configurations shown in FIGS. 5 and 7 described above.
[0048]
In FIG. 9B, the shape of the cell 115 is a polygon. Further, the sealing material 24 has an outer diameter d. ₃ Are common, but the inner circumference forms a polygon in accordance with the polygonal shape of the cell 115. In FIG. 9B, the width in the direction perpendicular to the direction of the flow path from the inlet port 116a to the outlet port 116b is formed smaller than the distance between the inlet port 116a and the outlet port 116b. This defines a slightly elongated cell shape in the direction of the flow path. Thereby, the residual amount of the chemical solution and the residual air at a position far away from the straight line 115c connecting the inlet port 116a and the outlet port 116b are reduced, so that the in-plane uniformity of various electrochemical reactions for measurement is improved. FIG. 9B shows an octagon as an example of a polygon. However, the present invention is not limited to this, and a polygon such as a triangle, a quadrangle, a pentagon, or the like may be used.
[0049]
FIG. 10A shows that the shape of the cell 115 is elliptical. The outer circumference of the sealing material 24 has an outer diameter d. ₃ , And the inner periphery has an elliptical shape. The center of the ellipse is O. The major axis of the ellipse is located on the straight line 115c, and the length of the major axis is 2 × d ₀ Is determined by The minor axis of the ellipse is located on a straight line 115e perpendicular to the straight line 115c and passing through the central axis O, and the length of the minor axis is 2 × d ₅ And d ₅ <D ₀ Holds. As described above, when the elliptical cell 115 is used, the width of the path of the chemical from the inlet port 116a to the outlet port 116b becomes smaller than that of the circular cell as shown in FIG. Along the shape. Therefore, the remaining amount of the chemical solution and the air remaining at the spread position of the path of the chemical solution are reduced, and the in-plane uniformity of various electrochemical reactions for measurement is improved.
[0050]
In FIG. 10B, the vicinity of the port on the outer periphery of the cross section of the cell 115 is defined by a curve expressed by a multi-order equation. The outer periphery of the seal member 24 has a circular shape with an outer diameter d3, and the inner periphery is determined by a curve expressed by a multi-order equation. The distance from the center O of the cell 115 to the intersection between the outer periphery of the cell 115 and the straight line 115c is d ₀ It is. More specifically, assuming that the intersection point between the outer periphery of the cell 115 and the straight line 115c is the origin, the straight line 115c intersects the y-axis and the straight line 115c perpendicularly, and the straight line 115f passing through the origin is the x-axis, The shape matches the shape of the curve of the given multi-order equation. The equation can be freely set, and can be set to, for example, a parabolic shape by setting.
[0051]
Note that this curve defines only the outer periphery of the half of the cell 115 on the side of the inlet port 116a. The curve of the same equation obtained by defining the x-axis as a straight line 115g indicates the outer peripheral shape of the cell on the side of the other half of the outlet port 116b. Is determined. Thereby, the outer peripheral shape of the cell 115 on the upstream side provided with the inlet port 116a and the downstream side provided with the outlet port 116b has a line-symmetrical shape with the straight line 115e as a boundary.
[0052]
In the example of FIG. 10B, the cross-sectional shape of the cell 115 near the inlet port 116a is a parabolic shape that extends toward the outlet port 116b. The cross-sectional shape of the cell 115 near the delivery port 116b is a parabolic shape that extends toward the delivery port 116a. As a result, when the area near the input port 116a and the output port 116b and deviated from the path connecting the input port 116a and the output port 116b, that is, the area at the port corner has a circular cross section as shown in FIG. It becomes narrower than. As a result, air and the chemical liquid hardly remain in the port corner region, and the current characteristics of the measurement are improved. 9B and 10A that the width of the cell 115, that is, the width in the direction perpendicular to the flow path is smaller than the distance between the inlet port 116a and the outlet port 116b. Is the same as
[0053]
FIG. 11A is a modified example of FIG. In the case of FIG. 10B, the case is shown where the outer peripheral shapes of the upstream side provided with the input port 116a and the downstream side 115 provided with the output port 116b are line-symmetric with each other. In the case of ()), the shape is asymmetric. Assuming that the intersection point between the outer periphery of the cell 115 and the straight line 115c is the origin, the straight line 115c is the y-axis, the straight line that intersects the straight line 115c perpendicularly, and the straight line passing through the origin is the 115f, the outer periphery of the cell 115 has a predetermined shape. It matches the shape of the curve of the multi-order equation.
[0054]
In the case of the figure, the equation is set to different values on the side of the sending port 116a and the side of the sending port 116b. As a result, as shown in the figure, cells having an asymmetric shape on the upstream side and the downstream side of the chemical solution with the straight line 115e as a boundary are formed. The delivery port 116b has a convex shape outside the cell 115, and the delivery port 116a has a slightly convex shape outside the cell 115, but its curvature is larger than that of the delivery port 116b.
[0055]
Of course, the shape of the cell 115 on the side of the sending port 116a and the side of the sending port 116b may be reversed from that shown in FIG. Further, as described later, a slight gap may be provided between the position where the delivery port 116b is formed and the position of the sealing material 24.
[0056]
Either the cell periphery on the upstream side or the downstream side may be in the shape of a curve expressed by a multi-order equation, the other may be in the shape of a curve not expressed by such an equation, or both may be expressed by a multi-order equation The shape of the curve may not be possible.
[0057]
FIG. 11B is a modified example of FIG. 9B. As in FIG. 9B, the outer peripheral shape of the cell 115 is a polygon, but in the case of FIG. 11B, it is a regular hexagon. Two vertices that are point-symmetric with respect to the center of the regular hexagon are located on the straight line 115c. The cell holes 115a and 115b are arranged so as to be in contact with the outer periphery of the regular hexagon near the two vertices. The point that the width of the cell 115, that is, the width in the direction perpendicular to the flow path is smaller than the distance between the inlet port 116a and the outlet port 116b is the same as in FIG. 9B and the like.
[0058]
9 (a), 9 (b), 10 (a), (b) and 11 (a), (b) described above, the outer peripheral positions of the sending port 116a and the sending port 116b are shown for reference. That is, the case where the outer peripheral positions of the cell holes 115a and 115b are in contact with the outer periphery of the cell 115 is described as an example, but the case where the outer periphery of the cell 115 with the input port 116a or the output port 116b fits inside the cell 115, The present invention is also applicable to a case where the counterbore is protruded from the outside (for example, a case where the counterbore hole 115d is used).
[0059]
FIGS. 12 to 16 show cross-sectional views of modified examples of the positional relationship between the cell 115 and the input port 116a and the output port 116b. The positions of the inlet port 116a and the outlet port 116b in FIGS. 12 to 16 indicate the outer peripheral positions of these ports in the cell holes 115a and 115b, that is, the positions of the openings for the cells 115. When used, it indicates the outer peripheral position of the cross section of the counterbore hole 115d.
[0060]
The positional relationship is different between the sending side and the sending side, and when the outer circumference of the cross section of the cell 115 is overlapped / contacted / separated from the sending port 116a or sending port 116b, the sending port 116a and the sending port 116b. Are symmetrical / asymmetrical with respect to the central axis O of the cell 115, and at least 18 types of combinations are conceivable.
[0061]
FIGS. 12 to 16 illustrate a case where the cross-sectional shape of the cell 115 is circular, as in the case illustrated in FIGS. 5, 7, and 9 (a). 10 (a), 10 (b), 11 (a), and 11 (b), the same applies only by changing the cell shape.
[0062]
FIG. 12A shows a case where the outer periphery of the port overlaps the outer periphery of the cross section of the cell 115 on both the sending side and the sending side. As in FIG. 7B, d ₀ Is the distance from the center O of the circular cell 115 to the outer periphery, d ₃ Is the outer diameter of the sealing material 24, d ₁ , D ₂ The radius of the port section (section of the cell holes 115a and 115b) of the inlet port 116a and the outlet port 116b, and the central axes of the inlet port 116a and the outlet port 116b ₁ And O ₂ And axis O and axis O ₁ Distance of d ₀₁ , Axis O and axis O ₂ Distance of d ₀₂ Then d ₀₁ + D ₁ > D ₀ , D ₀₂ + D ₂ > D ₀ Holds.
[0063]
FIG. 12B illustrates a case where the port side overlaps with the outer circumference of the cell 115 cross section on the sending side and the port outer circumference contacts with the outer circumference of the cell 115 cross section on the sending side. In this example, d ₀₁ + D ₁ > D ₀ , D ₀₂ + D ₂ = D ₀ Holds.
[0064]
FIG. 13A shows the case where the port side contacts the outer circumference of the cross section of the cell 115 on the sending side, and the outer circumference of the port overlaps the outer circumference of the cross section of the cell 115 on the sending side. The case where the configuration on the sending side and the configuration on the sending side are reversed is shown. In this example, d ₀₁ + D ₁ = D ₀ , D ₀₂ + D ₂ > D ₀ Holds.
[0065]
FIG. 13B shows a case where the outer circumference of the cross section of the cell 115 and the outer circumference of the port overlap on the sending side, and the outer circumference of the cross section of the cell 115 and the outer circumference of the port are separated on the sending side. In this example, d ₀₁ + D ₁ > D ₀ , D ₀₂ + D ₂ <D ₀ Holds.
[0066]
FIG. 14A shows a case where the outer periphery of the cross section of the cell 115 and the outer periphery of the port are separated on the sending side, and the outer periphery of the cross section of the cell 115 and the outer periphery of the port overlap on the sending side. In this example, d ₀₁ + D ₁ <D ₀ , D ₀₂ + D ₂ > D ₀ Holds.
[0067]
FIG. 14B shows a case where the outer periphery of the cross section of the cell 115 contacts the outer periphery of the port on the sending side, and the outer periphery of the port contacts the outer periphery of the cross section of the cell 115 on the sending side. In this example, d ₀₁ + D ₁ = D ₀ , D ₀₂ + D ₂ = D ₀ Holds. This example is common to the case shown in FIG.
[0068]
FIG. 15A shows a case where the outer periphery of the cross section of the cell 115 is in contact with the outer periphery of the port on the sending side, and the outer periphery of the port is separated from the outer periphery of the cross section of the cell 115 on the sending side. In this example, d ₀₁ + D ₁ = D ₀ , D ₀₂ + D ₂ <D ₀ Holds.
[0069]
FIG. 15B shows a case where the outer periphery of the cross section of the cell 115 is separated from the outer periphery of the port on the sending side, and the outer periphery of the cross section of the cell 115 is contacted with the outer periphery of the port on the sending side. In this example, d ₀₁ + D ₁ <D ₀ , D ₀₂ + D ₂ = D ₀ Holds.
[0070]
FIG. 16A shows a case where the outer periphery of the cross section of the cell 115 is separated from the outer periphery of the port on the sending side, and the outer periphery of the port is separated from the outer periphery of the cross section of the cell 115 on the sending side. In this example, d ₀₁ + D ₁ <D ₀ , D ₀₂ + D ₂ <D ₀ Holds.
[0071]
As described above, FIGS. 12 to 15 and FIG. 16A show nine modified examples depending on whether or not the port and the cell overlap. FIG. 16 (b) can be applied in further combination to these nine modifications depending on whether or not the port and the cell overlap. These nine modifications show the case where the input port 116a and the output port 116b are located symmetrically with respect to the center O of the cell 115. FIG. 16B shows the case where the input port 116a and the output port 116b are arranged. Is an asymmetric position with respect to the center O of the cell 115. Therefore, at least 18 combinations can be realized by further combining the nine modifications with FIG. 16B.
[0072]
As shown in FIG. 16B, a straight line connecting the input port 116a and the output port 116b does not pass through the central axis O of the cell 115. That is, the center O of the inlet port 116a ₁ A straight line 115h connecting the center O of the cell 115 to the center O of the delivery port 116b ₁ Does not coincide with the straight line 115c connecting the center O of the Therefore, the sending port 116a and the sending port 116b are provided at positions asymmetric with respect to the center O of the cell 115.
[0073]
In FIG. 16 (b), the outer periphery of the port overlaps the outer periphery of the cross section of the cell 115 on the liquid sending side, and the outer periphery of the port contacts the outer periphery of the cross section of the cell 115 on the discharge side, that is, d. ₀₁ + D ₁ > D ₀ , D ₀₂ + D ₂ = D ₀ (Corresponding to FIG. 12B), but the positional relationships shown in the remaining eight modifications shown in FIGS. 12 to 15 and 16A except for FIG. Of course, it may be applied.
[0074]
FIGS. 36 (a) and 36 (b) show the optimal shapes among the combinations of the shapes of the cells 115 shown in FIGS. FIG. 36A shows an example in which the cell shape in FIG. 11A is combined with the positional relationship between the ports in FIG. 13A, and FIG. 36B shows the cell shape in FIG. This is an example in which the port positional relationships in FIG.
[0075]
As shown in FIGS. 36 (a) and (b), when considering the flow path from the input port 116a to the output port 116b, an elongated cell shape in which the width of the flow path is narrower than the distance between each port. It has become. This reduces the residual amount of the chemical solution and the residual air at a point far away from the straight line connecting the inlet port 116a and the outlet port 116b, and improves the in-plane uniformity of the electrochemical reaction for measurement.
[0076]
Further, the area of the port corner of the input port 116a is narrow, and the curvature of the curve defining the cell edge near the port corner is larger than the curvature of the curve defining the cell edge near the port corner of the output port 116b. Further, the peripheral portion of the inlet port 116a is formed so as to contact or overlap with the peripheral portion on the upper surface of the cell 115. Further, the peripheral portion of the delivery port 116b is formed apart from the peripheral portion of the upper surface of the cell 115. Thereby, it is possible to reduce the residual chemical solution and air remaining near the port corner of the inlet port 116a when the chemical solution or the air is supplied, and to reduce the amount of the chemical solution or air generated at the port corner of the delivery port 116b when the chemical solution or the air is delivered. Fluctuations in the liquid velocity can be reduced, and residual air and the like can be reduced.
[0077]
Next, a method for manufacturing the above-described base sequence detection chip 21 and printed circuit board 22 will be described with reference to the process cross-sectional views of FIGS.
[0078]
After cleaning the silicon substrate 211, the silicon substrate 211 is heated to form a thermal oxide film 212 on the surface of the silicon substrate 211. A glass substrate may be used instead of the silicon substrate 211.
[0079]
Next, a Ti film 213 is formed with a thickness of, for example, 50 nm on the entire surface of the substrate, and then an Au film 214 is formed with a thickness of, for example, 200 nm by sputtering. Here, the Au film 214 preferably has a crystal plane orientation of <111> orientation. Next, the photoresist film 210 is patterned so as to protect a region to be an electrode or a wiring later (FIG. 17A), and the Au film 214 and the Ti film 213 are etched (FIG. 17B). In this embodiment, KI / I is used for etching the Au film 214. ₂ The mixed solution is mixed with NH for Ti etching. ₄ OH / H ₂ O ₂ A mixed solution was used. The etching of the Au film 214 includes a method using diluted aqua regia and a method of removing it by ion milling. Similarly, for the etching of the Ti film 213, a wet etching process using hydrofluoric acid or buffered hydrofluoric acid, for example, CF ₄ / O ₂ A method by dry etching using plasma with a mixed gas is also applicable.
[0080]
Next, the photoresist film 210 is removed by oxygen ashing (FIG. 17C). The step of removing the photoresist film 210 can be performed using a solvent, using a resist stripper, or using these together with an oxygen ashing step.
[0081]
Next, a photoresist film 215 is applied on the entire surface and patterned so as to open the electrode portion and the bonding pad (FIG. 17D). Then, hard baking is performed in a clean oven at, for example, 200 ° C. for 30 minutes. For the hard baking method, a hot plate can be used, and processing conditions can be appropriately changed. Here, the photoresist film 215 is selected as the protective film, but an organic film such as polyimide or BCB (benzocyclobutene) may be used instead of the photoresist. In addition, SiO, SiO ₂ An inorganic film such as SiN or SiN may be used for the protective film. In this case, the photoresist film 215 is opened to protect the electrode portion, SiO or the like is deposited, and a region other than the electrode portion is protected by a lift-off method, or SiN or the like is formed on the entire surface. The photoresist film 215 may be formed by forming a pattern so that only the portions are opened, removing the SiN film or the like on the electrode by etching, and finally peeling off the photoresist film 215.
[0082]
Next, chips are formed by dicing. Finally, to clean the surface of the electrode, CF ₄ / O ₂ A process using mixed plasma is performed. Thereby, the base sequence detection chip 21 is obtained. Then, the base sequence detection chip 21 is mounted on the printed circuit board 22 on which the electric connector 22a is mounted. Then, the bonding pads of the base sequence detection chip 21 and the lead wires on the printed circuit board 22 are connected by wire bonding. After that, the wire bonding portion is protected by using the sealing resin 23.
[0083]
Through the above steps, the printed circuit board 22 on which the base sequence detection chip 21 is mounted can be manufactured.
[0084]
FIG. 18 shows a top view of the prepared base sequence detection chip 21. As shown in FIG. 18, a plurality of electrodes 21a are provided near the center of the chip surface. The square electrode 21a in FIG. 18 is an electrode used as a working electrode and a counter electrode, and a cross-shaped electrode 21b that intersects the vertical and horizontal directions substantially vertically so as to divide the set of the working electrode and the counter electrode into approximately four. Is an electrode used as a reference electrode. The region where the electrodes 21a and 21b are formed is used so as to overlap the bottom surface of the cell 115 indicated by the broken line. Further, a bonding pad 221 is arranged around the chip. Each of the electrodes 21a and the electrode 21b are connected to the bonding pad 221 by a wiring 222. Although not shown in FIG. 18, the peripheral portion where the bonding pad 221 is formed is sealed with the sealing resin 23 described above.
[0085]
Next, an example of a specific configuration of the liquid feeding system 13 will be described with reference to FIG. The liquid feeding system 13 is roughly divided into a supply system provided on the channel 114a side of the cassette 11 and a discharge system provided on the channel 114b side.
[0086]
An air supply source 401 is connected to the uppermost stream of the pipe 404. On the downstream side of the air supply source 401, a check valve 402 for preventing a chemical solution other than air from flowing back to the air supply source 401 via a pipe 404 is provided, and further on the downstream side, a two-way solenoid valve is provided. 403 (V _a ) Is provided. Thus, the flow rate of the air flowing from the pipe 404 toward the chip cartridge 11 is controlled.
[0087]
A milli-Q water supply source 411 containing milli-Q water as one of the chemicals is connected to the pipe 414. On the downstream side of the milli-Q water supply source 411, there is provided a check valve 412 for preventing a chemical solution or air other than the milli-Q water from flowing back to the milli-Q water supply source 411. Solenoid valve 413 (V _wa ) Is provided. The communication between the pipe 404 and the pipe 415 and the communication between the pipe 414 and the pipe 415 are switched by the three-way solenoid valve 413. That is, the pipe 404 communicates with the pipe 415 when the three-way solenoid valve 413 is not energized, and the pipe 414 communicates with the pipe 415 when energized. Thereby, the supply of air and milli-Q water to the pipe 415 can be switched.
[0088]
A buffer supply source 421 containing a buffer (buffer solution) as one of the chemicals is connected to the pipe 424. On the downstream side of the buffer supply source 421, there is provided a check valve 422 for preventing a chemical solution or air other than the buffer from flowing back to the buffer supply source 421, and further on the downstream side, a three-way solenoid valve 423 (V _ba ) Is provided. The communication between the pipe 424 and the pipe 425 and the communication between the pipe 415 and the pipe 425 are switched by the three-way solenoid valve 423. That is, when the three-way solenoid valve 423 is not energized, the pipe 415 is connected to the pipe 425, and when energized, the pipe 424 is connected to the pipe 425. Thereby, the supply of the buffer to the pipe 425 and the supply of the air or the milli-Q water can be switched.
[0089]
The pipe 434 is connected to an intercalating agent supply source 431 containing an intercalating agent as one of the chemicals. A check valve 432 is provided on the downstream side of the intercalating agent supply source 431 to prevent a chemical solution or air other than the intercalating agent from flowing back to the intercalating agent supply source 431, and further on the downstream side, a three-way solenoid valve is provided. 433 (V _in ) Is provided. The three-way solenoid valve 433 switches between the communication between the pipe 434 and the pipe 435 and the communication between the pipe 425 and the pipe 435. That is, when the three-way solenoid valve 433 is not energized, the pipe 425 communicates with the pipe 435, and when energized, the pipe 434 communicates with the pipe 435. Thereby, the supply of the intercalating agent to the pipe 435 and the supply of air, milli-Q water or a buffer can be switched.
[0090]
As described above, by controlling the two-way solenoid valve 403 and the three-way solenoid valves 413, 423, and 433 in the air or chemical supply system, the air supplied to the chip cartridge 11 via the pipe 435, the milli-Q water, The supply of chemicals such as a buffer and an insert can be switched, and the supplied air and the flow rates of these chemicals can be controlled.
[0091]
The upstream side of the pipe 435 communicates with the above-described three-way solenoid valve 433, and the downstream side thereof communicates with the three-way solenoid valve 441 (V _cbin ) Are in communication. With the three-way solenoid valve 441, the pipe 435 can be branched into the pipe 440 and the bypass pipe 446. The three-way solenoid valve 441 switches the pipe 435 to communicate with the bypass pipe 446 when not energized, and switches the pipe 435 to communicate with the pipe 440 when energized. The three-way solenoid valve 445 switches the bypass pipe 446 to communicate with the pipe 450 when not energized, and switches the pipe 440 to communicate with the pipe 450 when energized. By these three-way solenoid valves 441 and 445, the supply of various chemicals and air can be switched to the bypass pipe 446 and the pipe 440.
[0092]
In the pipe 440, the two-way solenoid valve 442 (V _1in ), Chip cartridge 11, liquid sensor 443, two-way solenoid valve 444 (V _{1 out} ) 3-way solenoid valve 445 (V _cbout ) Is provided. The two-way solenoid valve 442 communicates with a flow path 114a corresponding to the feeding system of the chip cartridge 11, and the two-way solenoid valve 444 communicates with a flow path 114b corresponding to the sending system of the chip cartridge 11. ing. As a result, a chemical solution, air, and the like are supplied to the feed system of the chip cartridge 11 via the pipe 440, and the chemical solution, air, and the like can be discharged from the feed system of the chip cartridge 11. In addition, the two-way solenoid valves 442 and 444 can control the flow rate of a chemical solution, air, and the like in the liquid supply and discharge paths. The liquid sensor 443 can monitor the flow rate of the chemical solution flowing into the chip cartridge 11 or discharged from the chip cartridge 11.
[0093]
In the pipe 450, the two-way solenoid valve 451 (V _vin ), Decompression region 452, two-way solenoid valve 453 (V _out ), Liquid sending pump 454, three-way solenoid valve 455 (V _ww ) Is provided. The two-way solenoid valves 451 and 453 prevent backflow of the chemical solution and air in the path before and after the pressure reduction region 452. The liquid feed pump 454 is formed of a tube pump, and is characterized in that it is provided in a discharge system on the sending side (downstream side) when viewed from the chip cartridge 11. That is, by using a tube pump, the chemical solution does not come into contact with a mechanism other than the tube wall, which is preferable from the viewpoint of preventing contamination. Further, by supplying and discharging the chemical solution and the air to and from the chip cartridge 11 by the suction operation, the replacement of the chemical solution and the air inside the chip cartridge 11 can be performed not only in a lubricating manner, but also in the case of the piping. When the chip cartridge 11 is loosened or the chip cartridge 11 comes off the pipe 440, no liquid leakage occurs. Thereby, the safety of installation of the device is improved.
[0094]
Of course, a pump may be provided in the pipe on the upstream side of the chip cartridge 11, and the pump and the air may be pushed out to the chip cartridge 11 by the pump. Further, the pump is not limited to the tube pump, but may be a syringe pump, a plunger pump, a diaphragm pump, a magnet pump, or the like.
[0095]
The three-way solenoid valve 455 switches so that the pipe 450 communicates with the pipe 461 when not energized, and connects the pipe 450 with the pipe 463 when energized. The pipe 461 is provided with a waste liquid tank 462, and the pipe 463 is provided with an intercalating agent waste liquid tank 464. As a result, chemicals such as milli-Q water and buffer other than the intercalating agent can be guided to the waste liquid tank 462 by switching the three-way solenoid valve 455, and the intercalating agent can be guided to the intercalating agent waste liquid tank 464. This makes it possible to separate and collect the intercalating agent.
[0096]
The electromagnetic valves may be connected to each other by a pipe such as a Teflon tube, but in the present embodiment, the electromagnetic valves and the flow paths are integrated with the chip cartridge 11 on the upstream side and the downstream side, respectively. It has a manifold structure. As a result, the volume in the pipe is reduced, so that the required amount of the chemical solution can be significantly reduced. Further, since the flow of the chemical solution in the pipe is stabilized, the reproducibility and stability of the detection result are improved.
[0097]
A liquid sending step for detecting a base sequence using the liquid sending system 13 shown in FIG. 19 will be described with reference to the flowchart in FIG.
[0098]
First, a hybridization reaction between the DNA probe immobilized on the electrode 21a and the sample is performed in the cell 115 (s21). In the execution of the hybridization reaction, the temperature control mechanism 14 is controlled so that, for example, the bottom surface of the chip cartridge 11, that is, the bottom surface of the printed circuit board 22 is about 45 ° C., and is held, for example, for 60 minutes.
[0099]
In parallel with this hybridization reaction, a chemical solution line is set up (s22). Specifically, by controlling the three-way solenoid valves 441 and 445, the bypass pipe 446 is used, and the three-way solenoid valve 433 is energized to supply the intercalating agent from the intercalating agent supply source 431 for, for example, about 10 seconds. . The three-way solenoid valve 455 is energized, and the intercalating agent from the pipe 450 is stored in the intercalating agent waste liquid tank 464. Next, the intercalating agent and air are alternately introduced into the bypass pipe 446 from the pipe 435 alternately, for example, about every 5 seconds. Next, only air is introduced from the pipe 435 to the bypass pipe 446. At this stage, the waste liquid is switched to the waste liquid tank 462. Then, the buffer is introduced from the buffer supply source 421 into the bypass pipe 446. Thereafter, Milli-Q water and air are alternately introduced into the bypass pipe 446 from the pipe 435 alternately, for example, about every 5 seconds.
[0100]
When the startup of the chemical solution line is completed and the hybridization reaction is completed, the inside of the pipe is washed (s23). In the cleaning of the inside of the pipe, for example, after the temperature of the printed circuit board 22 is set to about 25 ° C. by the temperature control mechanism 14, the bypass pipe 446 is purged with milli-Q water, and then air and milli-Q water are alternately changed, for example, for about 5 seconds each. Introduce repeatedly. Next, the inside of the chip cartridge is washed (s24). In the cleaning in the chip cartridge, the chemical solution introduction path is switched from the bypass pipe 446 to the pipe 440, and air and milli-Q water are alternately introduced into the pipe 440, for example, about every 5 seconds. Then, after confirming that the chip cartridge 11 is filled with water by the liquid sensor 443, the introduction path is switched to the bypass pipe 446.
[0101]
Next, a buffer purge in the pipe is performed (s25). In the buffer purge in the pipe, air is first introduced into the bypass pipe 446 so that the buffer and the milli-Q water are not mixed. Next, the air and the buffer are alternately introduced into the bypass pipe 446 alternately, for example, about every 5 seconds. Then, the liquid sensor 447 provided in the bypass pipe 446 confirms that the buffer has replaced the bypass pipe 446.
[0102]
Next, buffer injection into the chip cartridge is performed (s26). In the buffer injection in the chip cartridge, first, the pipe is switched from the bypass pipe 446 to the pipe 440, and the air and the buffer are alternately introduced into the chip cartridge 11 alternately, for example, about every 5 seconds.
[0103]
Next, a buffer is filled into the chip cartridge 11 (s27). In filling the buffer, the buffer is introduced into the chip cartridge 11 while monitoring the state in the chip cartridge 11 with the liquid sensor 443, and unnecessary samples are washed by leaving the buffer at, for example, 60 ° C. for 30 minutes (s28). After the unnecessary sample washing step, the pipe 440 is switched to the bypass pipe 446, and the inside of the pipe is washed by introducing milli-Q water (s29). In this cleaning in the pipe, air and milli-Q water are alternately and repeatedly introduced, for example, for about 5 seconds each.
[0104]
Next, the inside of the chip cartridge is washed (s30). In the cleaning in the chip cartridge, the bypass pipe 446 is switched to the chip cartridge 11, and air and water are alternately introduced repeatedly, for example, for about 5 seconds each. After that, the liquid cartridge 443 is switched to the bypass pipe 446 after confirming that the chip cartridge 11 is filled with the milli-Q water.
[0105]
Next, the measurement is started. In the measurement, first, an insertion agent purge in a pipe is performed (s31). In the in-pipe insertion agent purge, the waste liquid is switched to the insertion agent waste liquid tank 464 while introducing air into the bypass pipe 446. Next, after alternately supplying the air and the insert agent to the bypass pipe 446 alternately, for example, about every 5 seconds, the liquid sensor 447 detects whether the bypass pipe 446 has been replaced with the insert agent.
[0106]
Next, an insertion agent is injected into the chip cartridge 11 (s32). In this step, the air and the insert are alternately introduced repeatedly, for example, about every 5 seconds after switching from the bypass pipe 446 to the chip cartridge 11 side.
[0107]
Next, under the monitoring of the liquid sensor 443, the filling agent is filled into the chip cartridge 11 (s33). Thereafter, measurement is performed (s34).
[0108]
When the measurement is completed, milli-Q water is introduced into the bypass pipe 446, and then air and milli-Q water are alternately introduced, for example, for about 5 seconds each, and then replaced with air to clean the inside of the pipe (s35).
[0109]
Finally, the chip cartridge 11 is replaced from the bypass pipe 446, and air and milli-Q water are alternately introduced, for example, for about 5 seconds, and the inside of the chip cartridge 11 is further replaced with air to clean the inside of the chip cartridge (s36). ), A series of liquid feeding steps is completed.
[0110]
As described above, according to the process shown in FIG. 20 using the liquid feeding system 13 of FIG. 19, in order to efficiently perform the replacement of the chemical solution, the inside of the pipe is connected with the air such as chemical solution / air / chemical solution / air. The liquid can be sent by creating a sequence in which the chemical liquid flows alternately. By adopting such a liquid sending method, it is possible to minimize the mixing of the old chemical liquid and the new chemical liquid in the chemical liquid exchange. As a result, the transition state of the liquid exchange is reduced, and the reproducibility of the final electrochemical characteristics can be improved. Further, it is possible to shorten the liquid sending time and reduce the amount of the chemical solution by increasing the efficiency of the chemical solution exchange. In addition, since the concentration of the chemical solution in the reaction cell 115 can always be kept constant by such a chemical / air sequence liquid supply, the in-plane uniformity of the current characteristics, that is, the reliability of detection is improved.
[0111]
As a method of filling the cell 115 with the chemical solution, in order to cope with the case where air bubbles are mixed, the inside of the pipe 440 on the downstream side of the chip cartridge with the two-way electromagnetic valve 444 serving as the chip cartridge outlet valve closed. By reducing the pressure in the decompression state (by controlling the two-way solenoid valve 451 while the pump 454 is operated, the decompression area 452 is depressurized and then the two-way solenoid valve 453 is controlled to change the decompression state in the decompression area 452 Therefore, by opening the two-way solenoid valve 444, a chemical solution can be introduced into the chip cartridge reaction cell 115.
[0112]
The timing of the liquid transfer shown in FIG. 20 is merely an example, and can be variously changed according to the purpose, target, condition, and the like of the measurement.
[0113]
FIG. 21 is a diagram illustrating a specific configuration of the measurement system 12. The measurement system 12 shown in FIG. 21 performs the negative feedback of the voltage of the reference electrode 503 with respect to the input of the counter electrode 502, so that the desired Is a three-electrode type potentiostat 12a for applying a voltage of 5V.
[0114]
More specifically, the potentiostat 12a changes the voltage of the counter electrode 502 so that the voltage of the reference electrode 503 with respect to the working electrode 501 is set to a predetermined characteristic, and electrochemically reduces the oxidation current of the intercalating agent. Measure.
[0115]
The working electrode 501 is an electrode on which a DNA probe having a target complementary base sequence complementary to the target base sequence is immobilized, and is an electrode for detecting a reaction current in the cell 115. The counter electrode 502 is an electrode that applies a predetermined voltage to the working electrode 501 and supplies a current into the cell 115. The reference electrode 503 is an electrode for negatively feeding back its electrode voltage to the counter electrode 502 in order to control the voltage between the reference electrode 503 and the working electrode 501 to a predetermined voltage characteristic, whereby the voltage of the counter electrode 502 is controlled. In addition, highly accurate oxidation current detection can be performed without being affected by various detection conditions in the cell 115.
[0116]
A voltage pattern generation circuit 510 that generates a voltage pattern for detecting a current flowing between the electrodes is connected to an inverting amplifier 512 (OP) for controlling a reference voltage of a reference electrode 503 via a wiring 512b. _c ) Is connected to the inverting input terminal.
[0117]
The voltage pattern generation circuit 510 is a circuit that converts a digital signal input from the control mechanism 15 into an analog signal to generate a voltage pattern, and includes a DA converter.
[0118]
The wiring 512b has a resistor R _s Is connected. The non-inverting input terminal of the inverting amplifier 512 is grounded, and the output terminal is connected to the wiring 502a. The wiring 512b on the inverting input terminal side of the inverting amplifier 512 and the wiring 502a on the output terminal side are connected by a wiring 512a. This wiring 512a has a feedback resistor R _ff And switch SW _f Is provided.
[0119]
The wiring 502a is connected to the terminal C. The terminal C is connected to the counter electrode 502 on the base sequence detection chip 21. When a plurality of counter electrodes 502 are provided, a terminal C is connected to each of them in parallel. Thus, a voltage can be simultaneously applied to the plurality of counter electrodes 502 by one voltage pattern.
[0120]
A switch SW for performing on / off control of voltage application to the terminal C is provided on the wiring 502a. ₀ Is provided.
[0121]
The protection circuit 500 provided in the inverting amplifier 512 has a configuration in which an excessive voltage is not applied to the counter electrode 502. Therefore, when an excessive voltage is applied during the measurement and the solution is electrolyzed, it does not affect the detection of the oxidation current of the desired intercalating agent, and stable measurement can be performed.
[0122]
The terminal R is connected to the voltage follower amplifier 513 (OP _r ) Is connected to the non-inverting input terminal. The inverting input terminal of the voltage follower amplifier is short-circuited by the wiring 513b and the wiring 513a connected to the output terminal. A resistor R is connected to the wiring 513b. _f Are connected between the resistance of the wiring 512b and the intersection of the wiring 512a and the wiring 512b. Thus, the voltage obtained by feeding back the voltage of the reference electrode 503 is input to the inverting amplifier 512 in the voltage pattern generated by the voltage pattern generating circuit 510, and the voltage of the counter electrode 502 is changed based on the output obtained by inverting and amplifying such a voltage. Control.
[0123]
The terminal W is connected to the trans-impedance amplifier 511 (OP _w ) Is connected to the inverting input terminal. The non-inverting input terminal of the trans-impedance amplifier 511 is grounded, and the wiring 511b and the wiring 501a connected to the output terminal are connected by the wiring 511a. A resistor R is connected to the wiring 511a. _w Is provided. The voltage at the output terminal O of the transimpedance amplifier 511 is _w , The current is I _w Then V _w = I _w ・ R _w It becomes. The electrochemical signal obtained from the terminal O is output to the control mechanism 15. Since there are a plurality of working electrodes 501, a plurality of terminals W and terminals O are provided corresponding to each of the working electrodes 501. Outputs from the plurality of terminals O are switched by a signal switching unit described later, and are subjected to A / D conversion, so that electrochemical signals from each working electrode 501 can be obtained almost simultaneously as digital values. A circuit such as the trans-impedance amplifier 511 between the terminal W and the terminal O may be shared by a plurality of working electrodes 501. In this case, the wiring 501a may include a signal switching unit for switching the wiring from the plurality of terminals W.
[0124]
The effect of the measuring system 12 using the potentiostat 12a in FIG. 21 will be described in comparison with the case where a conventional potentiostat is used. FIG. 22 shows a conventional potentiostat. As shown in FIG. 22, the configuration of the conventional potentiostat 12a 'is substantially common to the potentiostat 12a shown in FIG. The difference is that the inverting amplifier 512 is not provided with the protection circuit 500. The voltage at the output terminal I of the voltage pattern generation circuit 510 is V _refin , The voltage at terminal C is V _c And the voltage at the terminal R is V _refout And Due to the negative feedback of the reference pole 503, V _refout = R _f / R _s ・ V _refin Holds.
[0125]
In this case, the voltage V _refin , Switch SW ₀ And SW _f Switch switching state, voltage Vc and voltage V _refout FIG. 23 shows an example of the voltage characteristics and the switch switching state of the potentiostat 12a, and FIG. 24 shows an example of the potentiostat 12a '.
[0126]
In FIG. 23, (a) shows the voltage V _refin (B) is a switch SW ₀ Switch switching state, (c) is a switch SW _f (D) is the voltage V _c (E) is the voltage V _refout FIG.
[0127]
In FIG. 24, (a) shows the voltage V _refin (B) is a switch SW ₀ (C) is the voltage V _c (D) is the voltage V _refout FIG.
[0128]
A measuring method in the conventional potentiostat 12a 'will be described with reference to FIG.
[0129]
For example, as shown in FIG. ₁ To t ₃ Until the time t ₄ A voltage pattern is generated by the voltage pattern generation circuit 510 so as to linearly decrease the voltage so that the voltage becomes zero. Then, for example, as shown in FIG. ₁ Time t after a predetermined time has elapsed since ₂ In the switch SW ₀ Is closed and a voltage is applied to the counter electrode 502. In this case, at the start of the measurement, ie, the switch SW ₀ Switch SW until is closed ₀ Is open.
[0130]
Since the gain of the inverting amplifier 512 is very large, the switch SW ₀ If a slight voltage is applied to the inverting input terminal of the inverting amplifier 512 before is turned on and the feedback loop is formed, the output of the inverting amplifier 512 is saturated. On the other hand, the voltage V _refin Is saturated even due to the input offset voltage of the inverting amplifier 512. In this case, it saturates to the opposite polarity of the input offset voltage.
[0131]
Thus, the output voltage of the inverting amplifier 512 is saturated up to the vicinity of the power supply voltage of the inverting amplifier 512. Therefore, the switch SW ₀ Is closed, an excessive voltage is applied to the counter electrode 502. This excess voltage corresponds to the hatched portion in FIG. This excessive voltage causes an unintended electrochemical reaction such as electrolysis in the solution in the cell 115. As a result, the measurement of the intended electrochemical reaction is adversely affected.
[0132]
In order to solve the inconvenience of the conventional potentiostat 12a ', the potentiostat 12a of the present embodiment uses the protection circuit 500. In the case of the potentiostat 12a of the present embodiment, before starting the measurement, that is, at the time t _a Before the initial state, the voltage V _refin 0V, switch SW _f Is closed and switch SW ₀ Is opened. First, time t _a Switch SW ₀ Is closed. In this state, the switch SW _f Is still open, and the protection circuit 500 is not functioning. Since the inverting amplifier 512 is always used with negative feedback applied, no excessive voltage is applied to the counter electrode 502.
[0133]
This time t _a Time t after a predetermined time from _b And switch SW _f Is closed, and the protection circuit 500 is operated. Then time t ₁ From the voltage V generated by the voltage pattern generation circuit 510 _refin Is applied. This voltage V _refin As a result, a desired voltage is set to the reference electrode 503, so that the response has a first-order lag characteristic, and no excessive voltage is applied to the counter electrode 502.
[0134]
FIG. 25 is a diagram showing a current / voltage characteristic curve applied to the counter electrode 502 in the potentiostats 12a and 12a '. As shown in FIG. 25, in the case of the conventional potentiostat 12a ', both the current and the voltage have characteristics of being greatly negative, whereas in the case of the potentiostat 12a of the present embodiment, the voltage is negative. Also, the current falls to a constant value. When the voltage becomes a negative value, unintended electrolysis in the solution of the cell 115 proceeds. As a result, for example, there are adverse effects such as generation of bubbles in the electrode and change in the composition of the electrode. On the other hand, by providing the protection circuit 500 as in the example of the present embodiment, it is possible to prevent an unintended voltage from being applied to the counter electrode 502. Can be avoided. Therefore, stable measurement is possible without affecting the detection of the oxidation current of the desired intercalating agent.
[0135]
Modifications of the potentiostat 12a as the measurement system 12 shown in FIG. 21 are shown in FIGS. FIGS. 26 and 27 show examples in which a three-electrode potentiostat is used as the measurement system 12, and FIGS. 28 and 29 show examples in which a four-electrode potentiostat is used as the measurement system 12.
[0136]
The potentiostat 12b shown in FIG. 26 has the same basic configuration as the potentiostat 12a shown in FIG. The same components are denoted by the same reference numerals, and detailed description will be omitted. The potentiostat 12b differs from the potentiostat 12a in that the protection circuit 500 including the wiring 512a is not provided. Instead of this protection circuit 500, a resistor R _c Is provided. Thus, the resistance R is connected in series with the output of the inverting amplifier 512 on the counter electrode 502 side. _c , The voltage applied to the counter electrode by the electric double layer capacitance has a first-order lag. Thereby, the influence on the chemical solution in the cell 115 can be reduced.
[0137]
The configuration of the potentiostat 12c shown in FIG. 27 is slightly different from that of the potentiostats 12a and 12b. In this potentiostat 12c, the current detection resistor R _c Is provided on the counter electrode 502 side, and the detected current is converted into a voltage by the high input impedance differential amplifier 520. Hereinafter, the configuration will be described in more detail.
[0138]
As shown in FIG. 27, a voltage pattern generating circuit 510 for generating a voltage pattern for detecting a current flowing between the electrodes is connected to an inverting amplifier 512 (OP) via a wiring 512b. _c ) Is connected to the inverting input terminal. This wiring 512b has a resistor R _s Is connected. The non-inverting input terminal of the inverting amplifier 512 is grounded, and the output terminal is connected to the wiring 512f. The output terminal and the inverting input terminal of the inverting amplifier 512 are connected by the protection circuit 500.
[0139]
The wiring 512f is a switch SW for performing on / off control of voltage application to the terminal C. ₀ Is provided. The wiring 512f branches into two wirings 521a and 521b at an intersection 512c. The wiring 521a is connected to the non-inverting input terminal of the amplifier 522 of the high input impedance differential amplifier 520.
[0140]
The wiring 521b has a current detection resistor R _c Is provided. Further, the wiring 521b branches into a wiring 502a and a wiring 521e at an intersection 521d. The wiring 502a is connected to the terminal C, and the wiring 521e is connected to the non-inverting input terminal of the amplifier 523 in the high input impedance differential amplifier 520.
[0141]
A voltage follower amplifier 513 for feeding back a voltage from the terminal R on the reference pole 503 side to the inverting input terminal of the inverting amplifier 512, wirings 513a and 513b, and a resistor R _f Is common to FIG.
[0142]
The terminal W on the working electrode 501 side is grounded by the wiring 501a.
[0143]
The high input impedance differential amplifier 520 includes a current detection resistor R _c The output from the wiring 521a that does not pass through _c Is amplified and output to the terminal O. The inverting input terminal of each of the amplifiers 522 and 523 is a resistor R ₁ Are connected by a wiring 522a having The inverting input terminal and the output terminal of the amplifier 522 are connected to a resistor R. ₂ Are connected by a wiring 522b having The inverting input terminal and the output terminal of the amplifier 523 are connected to a resistor R. ₃ Are connected by a wiring 523a having The output of amplifier 522 is a resistor R ₄ To the inverting input terminal of the amplifier 524. The output of the amplifier 523 is a resistor R ₅ To the non-inverting input terminal of the amplifier 525. Amplifier 524 has a resistor R ₆ Grounded. The inverting input terminal and output terminal of the amplifier 524 are connected to a resistor R. ₇ Are connected by a wiring 522d having The output terminal of the amplifier 524 is connected to the terminal O by a wiring 524b.
[0144]
In the case of the potentiostat 12c, the oxidation current is detected not from the working electrode 501 but from the counter electrode 502 side.
[0145]
As described above, even when the potentiostat 12c as shown in FIG. 27 is used, the same effect as that of the potentiostat 12a can be obtained.
[0146]
The configuration of the counter electrode 502 side and the working electrode 501 side of the four-electrode type potentiostat 12d shown in FIG. 28 is common to the configuration of the potentiostat 12a of FIG. In the case of the potentiostat 12d, the voltage from the two reference poles 5031 and 5032 is differentially amplified using the high input impedance differential amplifier 520, and the output voltage is fed back to the inverting amplifier 512 on the counter electrode 502 side. As described above, the potential difference between the two reference electrodes is detected, and the supply current from the counter electrode 502 is controlled so that the value has a predetermined voltage characteristic.
[0147]
As shown in FIG. 28, the terminal R on the reference electrode 5031 side ₁ Is connected to the non-inverting input terminal of the amplifier 523. Terminal R on reference electrode 5032 side ₂ Is connected to the non-inverting input terminal of the amplifier 522. The high input impedance differential amplifier 520 differentially amplifies and outputs two voltages of the non-inverting input terminals of each of these amplifiers 522 and 523. The output side has a resistor R _f To the wiring 512b.
[0148]
As described above, even when the potentiostat 12d shown in FIG. 28 is used, the same effect as the potentiostat 12a can be obtained.
[0149]
The four-electrode potentiostat 12e shown in FIG. 29 has the same basic configuration as the potentiostat 12c shown in FIG. The difference from the potentiostat 12c is that the potentiostat 12e has two reference pole extraction voltages, and that the two voltages are differentially amplified and fed back to the counter electrode 502 side. Since the configurations on the counter electrode 502 side and the working electrode 501 side are common to the potentiostat 12c, detailed description will be omitted. 520 ′ is a high input impedance differential amplifier having the same configuration as the high input impedance differential amplifier 520 described above.
[0150]
As shown in FIG. 29, the potentiostat 12e has two terminals R on the side of the reference electrode 5031. ₁ And the terminal R on the reference electrode 5032 side ₂ Are connected to the non-inverting input terminal of the amplifier 523 and the non-inverting input terminal of the amplifier 522, respectively. As described above, the high input impedance differential amplifier 520 differentially amplifies these two inputs and outputs the result. The output side has a resistor R _f Is connected, and the resistance R _f Is connected to the wiring 512b. As a result, the output of the high input impedance differential amplifier 520 is fed back to the input side of the inverting amplifier 512.
[0151]
FIG. 30 is a conceptual diagram showing the relationship between the control mechanism 15 and other components of the computer 16. As shown in FIG. 30, the computer 16 includes a main processor 16a and an interface 16b. Data can be transmitted and received between the plurality of control mechanisms 15 via the local bus 17 via the interface 16b. The control mechanism includes a measurement control mechanism main body 15a and a data memory 15b for storing data handled by the measurement control mechanism main body 15a. One control mechanism 15 is provided for each of the measurement units 10. In this way, by connecting a plurality of connected measurement units 10 to one main processor 16a, the load on the main processor 16a can be reduced.
[0152]
FIG. 31 is a diagram illustrating an example of a detailed configuration of the control mechanism 15. As shown in FIG. 31, the measurement control mechanism main body 15a has an initial value register 151, a step value register 152, an end value register 153, an interval register 154, and an operation setting register 155 connected to the local bus 17.
[0153]
The initial value register 151, step value register 152, end value register 153, interval register 154, and operation setting register 155 store an initial value, step value, end value, measurement time interval, and operation mode that can be set by the main processor 16a, respectively. I do. When these initial value, step value, end value, measurement time interval, and operation mode are set, the data measurement operation is started.
[0154]
The initial value, the step value, and the end value indicate values corresponding to the voltage values of the voltage pattern generated by the voltage pattern generation circuit 510, and the voltage pattern is set as a digital value for each step value from the initial value to the end value. You. For example, time t ₁ From time t ₅ When a voltage pattern having a predetermined waveform is generated until time t ₁ Is equivalent to the initial value, and the time t ₁ , The voltage value fluctuates by the step value at every measurement time interval Δt, and such a voltage value continues to be stepped until the end value.
[0155]
The selector 158 selects and outputs the initial value only at the start of the measurement from the output value of the initial value register and the output value of the adder 156, and selects and outputs the addition result of the adder 156 from the next data. The output value of the selector 158 is output to the voltage pattern generation circuit 510 of the measurement system 12 in synchronization with the output signal from the timing generator 161. Voltage pattern generation circuit 510 generates a voltage having a voltage value corresponding to the output value from selector 158. As a result, a voltage pattern having the voltage waveform shown in FIG. 23A can be generated.
[0156]
The addition register 157 temporarily stores the output value of the selector 158 in synchronization with the output signal of the timing generator 161.
[0157]
The adder 156 adds the step value of the step value register 152 to the initial value of the initial value register 151, and outputs the result to the selector 158 and the comparator 159. Since the value stored in the addition register 157 corresponds to the voltage value output to the measurement system 12, the adder 156 calculates the value corresponding to the voltage value obtained by adding the step value to the output voltage value to the measurement system 12. Output. Comparator 159 compares the addition result of adder 156 with the end value from end value register 153, and outputs a signal indicating the end of counting to counter 160 when the addition result exceeds the end value.
[0158]
The counter 160 continues to count the clock until a signal is input from the comparator 159 to stop counting based on the operation setting mode from the operation setting register 155 for a time period determined by the measurement time interval from the interval register 154. . In the operation setting mode, for example, a single measurement mode, a four-pole setting mode, an eight-pole setting mode, or the like can be set according to the number of simultaneously measured working electrodes. For example, when the single measurement mode is set, the counter 160 counts for a time period determined by the measurement time interval, and outputs the count value to the timing generator 161. When the four-pole setting mode is set, counting is performed for each time period obtained by dividing the measurement time interval into four, and the count value is output to the timing generator 161. As described above, when the multi-pole setting mode is set, the measurement time interval is counted for each time period divided by the number of poles.
[0159]
The timing generator 161 outputs an address signal and a write signal to the data memory 15b in synchronization with the output timing of the count value from the counter 160 while counting clocks. Further, the timing generator 161 switches the signal switching unit 163 of the signal detection unit 162 according to the operation setting mode from the operation setting register 155.
[0160]
The signal switching unit 163 is connected to each of the terminals O of the plurality of working electrodes 501 of the measurement system 12. Although the electrochemical signals due to the intercalating agent can be simultaneously detected from the terminal O by the plurality of working electrodes 501, the electrochemical signals from the plurality of working electrodes 501 can be selectively detected by the signal switching unit 163.
[0161]
The signal detection unit 162 AD-converts the electrochemical signal from the working electrode 501 switched by the signal switching unit 163 controlled by the timing generator 161, and outputs the electrochemical signal to the data memory 15 b via the data bus 164. Thus, every time a write signal is input from the timing generator 161, data from the data bus 164 can be sequentially written to the data memory 15 b at an address position given for each write signal.
[0162]
For example, in the single-pole setting mode, if the measurement time interval is 10 msec, the write signal and one address are output from the timing generator 161 to the data memory 15 b once every 10 msec, and the signal detection unit 162 outputs the data bus. One digitally converted value of the electrochemical signal is output to the data memory 15b via 15b.
[0163]
In the case of the 4-pole setting mode, if the measurement time interval is 10 msec, the write signal and the four addresses are output from the timing generator 161 to the data memory 15b four times in 10 msec, and the signal detection unit 162 outputs the data bus 15b. , Four digitally converted values of the electrochemical signal are sequentially output to the data memory 15b. As a result, electrochemical signals detected almost simultaneously at each measurement time interval can be stored as data.
[0164]
In order to improve the accuracy of the measurement, in the case of the multi-pole setting mode, the timing of signal detection from the plurality of working electrodes 501 may be shortened without synchronizing with the timing obtained by dividing the measurement time interval into equal intervals. it can. For example, by generating a plurality of switching signals of the signal switching unit 163 at a short time within the measurement time interval, measurement accuracy independent of the measurement time interval can be maintained. For example, if the measurement time interval is 10 msec, the timing generator 161 does not generate the switching signal until the first 9 msec, generates four switching signals in 1 msec from 9 msec to 10 msec, and outputs it to the signal switching unit 163. Is programmed. Thus, the electrochemical signals from the four working electrodes 501 can be detected within 1 msec. Therefore, even if the measurement time interval is set to be long, the measurement time interval does not vary due to the measurement time interval, and high accuracy can be maintained.
[0165]
The measurement data stored in the data memory 15b is read by the main processor 16a of the computer 16 and used for various signal analysis.
[0166]
As described above, by selectively switching a plurality of measured electrochemical signals by the timing generator 161 in a shorter time than the measurement time interval, it is possible to measure each signal of the working electrode 501 almost simultaneously. it can.
[0167]
Next, an example of a measurement data analysis method in which a signal is analyzed by the computer 16 based on the measurement data will be described. Here, the analysis method of the type determination for determining whether the base at the SNP position of the target DNA is G type (homo type), T type (homo type), or GT type (hetero type) is described with reference to the flowchart of FIG. Will be explained. Although not particularly shown in FIG. 1 or FIG. 30, the main processor 16a of the computer 16 executes an analysis program including a plurality of instructions for performing type determination filtering, type determination processing, determination result output, and the like. Performs type determination filtering, type determination processing, and determination result output. Further, a control program is separately provided for controlling the control mechanism 15 described above. The analysis program and the control program may be executed by a recording medium reading device provided in the computer 16 reading the analysis program stored in the recording medium, or a storage device such as a magnetic disk provided in the computer 16. And may be executed by being read.
[0168]
As a precondition for performing this measurement data analysis, first, four types of target base sequences to be detected are prepared as bases at SNP positions A, G, C, and T, and a base sequence complementary to the target base sequence is prepared. A plurality of target complementary DNA probes of each type are immobilized on each working electrode 501. In addition, a plurality of DNA probes (hereinafter, referred to as negative controls) having base sequences different from those of the four types of target complementary DNA probes are immobilized on another working electrode 501 (s61). The number of types of DNA probes immobilized on the working electrode 501 is basically one.
[0169]
Next, a sample containing the sample DNA probe is injected into the base sequence detection chip having the target complementary DNA probe immobilized thereon to cause an electrochemical reaction such as a hybridization reaction (s62). Then, a representative current value is calculated by using the measurement system 12 after the introduction of (S63).
[0170]
The representative current value refers to a numerical value effective for quantitatively grasping the occurrence of a hybridization reaction of each DNA probe, and as an example, the maximum value (peak current value) of the detected signal current value is used. Applicable. The peak current value is calculated by measuring the oxidation current signal from the intercalating agent bound to the double-stranded DNA hybridized to the DNA probe immobilized on each working electrode 501, and obtaining the peak of the current value. Derived. It is desirable to detect the peak current value by subtracting a background current other than the oxidation current signal from the intercalating agent.
[0171]
Of course, any value may be determined as the representative current value according to the accuracy and purpose of the signal processing, but for example, an integrated value of the oxidation current signal or the like is applicable. Of course, not only the current value, but also a voltage value, a value obtained by performing a numerical analysis process on the current and the voltage, and the like can be determined as the representative value.
[0172]
The measured data on the target DNA having A, G, C, and T bases at the SNP position, that is, the representative current values are represented by X, respectively. _a , X _g , X _c , X _t And the representative current value of the negative control DNA probe is X _n Is defined. Also, since a plurality of representative current values can be obtained for each type, the first X value is used to identify each of them. _a To X _a1 X is the second Xa _a2 , ... are defined.
[0173]
In addition, the number of representative current values obtained from the target DNA having the A, G, C, and T bases at the SNP position is represented by n. _a , N _g , N _c , N _t , The number of representative current values obtained for the negative control is n _n Is defined as
[0174]
Next, the obtained representative current value X _a , X _g , X _c , X _t , X _n Among them, a type determination filtering process is executed to remove apparently abnormal data (s64).
[0175]
FIG. 33 shows a flowchart of the type determination filtering process. The type determination filtering process of FIG. _a , X _g , X _c , X _t , X _n Are performed separately. For example X _a For example, X _a N obtained for _a Among the representative current values, a representative current value that is apparently abnormal data is excluded by this type determination filtering. X _g , X _c , X _t , X _n Is performed similarly.
[0176]
In the description of FIG. 33, the same processing is performed according to the data type. _a An example of filtering will be described.
[0177]
Specifically, as shown in FIG. 33, first, all measurement data of the measurement group are set, that is, a data set is set (s81). For example X _a Then X _a1 , X _a2 , ..., X _ana Is set as a data set.
[0178]
Next, these measurement data X _a1 , X _a2 , ..., X _ana CV value (hereinafter referred to as CV ₀ ) Is calculated (s82). This CV ₀ Is the measurement data X _a1 , X _a2 , ..., X _ana Divided by the mean. And the obtained value CV ₀ Is 10%, that is, 0.1 or more (s83).
[0179]
If it is 10% or more, the CV value of na-1 data sets excluding the minimum value among the measurement data (hereinafter, CV value) ₁ ) Is calculated (s84). If it is less than 10%, it is determined that there is no clearly abnormal data, and the process proceeds to the type determination described later.
[0180]
CV ₁ Is calculated, then CV ₀ ≧ 2 × CV ₁ It is determined whether or not (s85). If this inequality holds, the process proceeds to (s86), further defines na-2 data sets other than the minimum value among the measured data as new data sets, returns to (s82), and performs filtering of abnormal data. Repeat.
[0181]
If the inequality expression does not hold, it is determined that abnormal data exists on the maximum value side instead of the minimum value side, and the CV values of na-2 data sets (hereinafter, CV values) of the measurement data excluding the maximum value are determined. ₂ ) Is calculated (s87). And CV ₀ ≧ 2 × CV ₂ Is determined (S88). If the condition is satisfied, na-3 data sets other than the maximum value among the measured data are newly defined as data sets, and the process returns to (s82) to repeat the filtering of abnormal data. If not, it is determined that there is no obviously abnormal data, and the process proceeds to the type determination described later.
[0182]
The type determination filtering shown above is _g , X _c , X _t , X _n Also do.
[0183]
Next, a type determination process is performed using the obtained type determination filtering result (s65). An example of this type determination processing will be described with reference to the flowchart in FIG. Note that the example of FIG. 34 shows a case of type determination for determining whether the base at the SNP position of the target DNA is G-type, T-type, or GT-type. This type determination processing is roughly divided into a maximum group determination algorithm and a two-sample t-test algorithm.
[0184]
As shown in FIG. 34, first, the average value of the representative current values for each group is extracted (s91). Group is X _a , X _g , X _c , X _t , X _n For example, those having different target nucleotide sequences belong to another group, and those having the same target nucleotide sequence belong to the same group. In (s64), the measurement data from which the abnormal data is clearly excluded by the type determination filtering is extracted. Of course, measurement data from which the above data has been eliminated may be extracted by filtering other than the type determination filtering in (s64), or measurement data without any filtering may be extracted. Note that, instead of the average value of the representative current values, another statistical processing value obtained by performing statistical processing from these statistical values may be obtained.
[0185]
The case where the base at the SNP position of the target DNA is A, G, C, T will be described as groups A to T, respectively, and the negative control will be described as group N. Further, the obtained average value is represented by X _a , X _g , X _c , X _t , X _n For each group, M _a , M _g , M _c , M _t , M _n And
[0186]
Next, the obtained average value M _a , M _g , M _c , M _t , M _n , The maximum is the average value M of group G _g It is determined whether or not (s92). If it is the maximum, the process proceeds to (s93), and if it is not the maximum, the process proceeds to (s97).
[0187]
In (s97), the average value M _a , M _g , M _c , M _t , M _n , The maximum is the average value M of the group T _t It is determined whether or not. If it is the maximum, the process proceeds to (s98). If it is not the maximum, both the groups G and T are not the maximum.
[0188]
In (s93), the measurement data X of the group G _g1 , X _g2 , ..., measurement data X of group N _n1 , X _n2 ,... Are determined. For example, a two-sample t-test is used to determine whether there is a difference. Specifically, a representative relationship between the probability P obtained by the two-sample T test and the significance level α is compared,
H0: If P ≧ α, no significant difference (null hypothesis)
H1: If P <α, there is a significant difference (alternative hypothesis)
Is determined. The significance level α can be set by the user using the computer 16. In the example of (s93), a question of H1 is raised as to whether there is a difference between the measurement data of the group G and the measurement data of the group N, and if there is no difference between these two groups in response to this question. A hypothesis H0 to be assumed is set. Then, the average value M of the measurement data of the group G _g And the average value M of the measurement data of group N _n , The difference between the two groups is summarized and the probability is determined. The calculation of the probability is based on the statistical value X of the group G. _g1 , X _g2 , ... and the statistical value X of group N _n1 , X _n2 ,... Are calculated, and the probability P is obtained from the integral value of the probability density variable of the t distribution.
[0189]
Regarding the obtained probability P, if P ≧ α, H0 cannot be rejected and the determination is suspended. That is, it is determined that there is no difference. If P <α, H0 is rejected, hypothesis H1 is adopted, and it is determined that there is a difference.
[0190]
In this way, when the determination result is determined to be “difference”, the process proceeds to (s94), and when the determination result is “no difference”, the determination is re-tested as impossible.
[0191]
In (s94), it is determined whether or not there is a difference between the two groups using the same two-sample t-test as in (s93) for group G and group A. If there is a difference, the process proceeds to (s95).
[0192]
In (s95), it is determined whether or not there is a difference between the two groups using the same two-sample t-test as in (s93) for group G and group C. If there is a difference, the process proceeds to (s96), and if there is no difference, it is re-tested as impossible to determine.
[0193]
In (s96), it is determined whether or not there is a difference between the two groups using the same two-sample t-test as in (s93) for group G and group T. If there is a difference, it is determined to be a group G type. This is because the group G type has the maximum average value and a difference from the other measurement groups. If there is no difference, it is determined to be a group GT type. This is because the group G type has the maximum average value, but there is no difference in the measurement result between the group G type and the group T type.
[0194]
In (s98), it is determined whether or not there is a difference between the two groups using the same two-sample t-test as in (s93) for group T and group N. If there is a difference, the process proceeds to (s99).
[0195]
In (s99), it is determined whether or not there is a difference between the two groups for the group T and the group A using the same two-sample t-test as in (s93). If there is a difference, the process proceeds to (s100).
[0196]
In (s100), it is determined whether or not there is a difference between the two groups using the same two-sample t-test as in (s93) for groups T and C. If there is a difference, the process proceeds to (s101).
[0197]
In (s101), it is determined whether or not there is a difference between the two groups using the same two-sample t-test as in (s93) for groups T and G. If there is a difference, it is determined to be a group T type. This is because the group T type has the maximum average value and a difference from the other measurement groups. If there is no difference, it is determined to be a group GT type. This is because the group T type has the maximum average value, but there is no difference in the measurement results between the group T type and the group G type.
[0198]
The above determination result is displayed on a display device (not shown) provided in the computer 16 (s66). By using such a type determination algorithm, it is possible to determine a hetero type.
[0199]
FIGS. 32 to 34 show a method of determining which of the G type, the T type and the GT type, but any two of the A type, the G type, the C type, and the T type. It is needless to say that the present invention can be applied to the determination of types or their heterogeneity. Further, it is not always necessary to acquire measurement data for four types of A, G, C, and T groups, and it is sufficient to acquire only two groups for two possible bases of SNP. One group of the negative control may be added to the two groups.
[0200]
An automatic base sequence analysis method using the above-described base sequence detection apparatus will be described with reference to the sequence diagram of FIG.
[0201]
As shown in FIG. 35, first, automatic analysis condition parameters for automatic analysis are set using the computer 16, and the user instructs the computer 16 to execute automatic analysis based on the set automatic analysis condition parameters (s301). ). The automatic analysis condition parameters are control parameters for controlling the control mechanism 15. The control parameters used in the control mechanism 15 include a measurement system control parameter for controlling the measurement system 12, a liquid supply system control parameter for controlling the liquid supply system 13, and a temperature control mechanism for controlling the temperature control mechanism 14. Consists of control parameters.
[0202]
The measurement system control parameters are input setting parameters stored in the above-described initial value register 151, step value register 152, end value register 153, interval register 154, and operation setting register 155 shown in FIG. , End value, measurement time interval, and operation mode.
[0203]
The liquid supply system control parameters control the electromagnetic valves 403, 413, 423, 433, 441, 442, 444, 445, 451, 453, and 463 shown in FIG. 19, and control the liquid sensors 443 and 447. It has a sensor control parameter and a pump control parameter for controlling the pump 454. These electromagnetic valve control parameters, sensor control parameters, and pump control parameters are used as conditions for sequentially executing a series of steps as shown in (s22) to (s36) in FIG. Control timing, control conditions for controlling the control target, and the like are included as details of the parameters.
[0204]
The temperature control parameter is given in principle along with the liquid sending system control parameter. That is, by setting the liquid sending system control parameters, the temperature control parameters are set corresponding to the operation of the liquid sending system 13. Thereby, the temperature control of the temperature control mechanism 14 linked with the liquid feeding system 13 can be performed.
[0205]
By executing the automatic analysis, the automatic analysis condition parameters are transmitted to the control mechanism 15 (s302). The control mechanism 15 controls the measurement system 12 based on the measurement system control parameter among the received automatic analysis condition parameters, controls the liquid supply system 13 based on the liquid supply system control parameter, and controls the temperature based on the temperature control mechanism control parameter. The control mechanism 14 is controlled. Further, the control mechanism 15 manages the timing for controlling the measurement system 12, the liquid feeding system 13, and the temperature control mechanism 14 based on control timing and control conditions included in each control parameter. Therefore, the control sequence can be freely determined by the automatic analysis condition parameters set by the user. In FIG. 35, a typical example will be described.
[0206]
In addition to this automatic analysis, the user prepares the chip cartridge 11. First, a printed circuit board 22 in which a base sequence detection chip 21 in which a desired DNA probe is fixed to a working electrode 501 is sealed is fixed to a support 111 of the chip cartridge 11 by a board fixing screw 25, Is mounted (s401). Then, with the sealing material 24 placed at a predetermined position on the base sequence detection chip 21, the chip cartridge upper lid 112 and the support 111 are fixed by the upper lid fixing screw 117, and the cell 115 is prepared in a state where it is formed. (S402). A sample is injected into the chip cartridge 11 from the sample injection port 119 (s403). The hybridization reaction (s21) is started by mounting the chip cartridge 11 on the apparatus main body and performing a start operation. It is desirable that the volume of the sample to be injected is slightly larger than the volume of the cell 115. As a result, the cell 115 can be completely filled with the sample without remaining air.
[0207]
The control mechanism 15 starts controlling the timing of the measurement system based on the measurement system control parameters received from the computer 16 (s303).
[0208]
Further, the control mechanism 15 sequentially controls each component of the liquid sending system 13 based on the liquid sending system control parameters received from the computer 16 (s304). Although not particularly shown in FIG. 35, the temperature of the temperature control mechanism 14 is controlled based on the temperature control mechanism control parameters in conjunction with the control of the liquid supply system 13. With this control, the liquid sending system 13 automatically executes the liquid sending step including the hybridization reaction shown in (s21) to (s36) (excluding s34) in FIG. 20 (s305), and specifies the liquid sending step in the liquid sending step. The temperature control mechanism 14 is automatically controlled so that the base sequence detection chip 21 is set at the set temperature.
[0209]
The control mechanism 15 issues a measurement command to the measurement system 12 in synchronization with the timing of the measurement step (s34) in the middle of the liquid sending step (s305). That is, at the timing of the measurement step (s34) of the liquid sending step, the initial value, step value, step value register 152, end value register 153, interval register 154, and operation setting register 155 of the control mechanism 15 are stored in the initial value, step value, Store the end value, measurement time interval, and operation setting mode. The measurement system timing control in (s303) described above may be performed simultaneously with (s305).
[0210]
The measurement system 12 performs measurement by generating, for example, a voltage pattern based on the measurement command (s306), and the obtained measurement signal is output from the terminal O to the control mechanism 15 (s307). The control mechanism 15 performs signal processing on the received measurement signal and stores it in the data memory 15b as measurement data (s308). This measurement data is output to the computer 16 via the local bus 17 (s309). The computer 16 receives this measurement data (s310).
[0211]
When the necessary measurement data is obtained in this way, the computer 16 executes the type determination filtering (s64) shown in FIG. 33 based on the measurement data. When the type determination filtering is completed, the type determination process shown in FIG. 34 is executed based on the filtered data (s65). Finally, the obtained judgment processing result is displayed on a display device provided in the computer 16 (s66).
[0212]
As described above, according to the present embodiment, since the shape of the cell 115 is optimized, in-plane uniformity in the cell 115 can be improved.
[0213]
After injecting the sample DNA solution into the chip cartridge 11, from hybridization, washing of nonspecifically adsorbed DNA with a buffer solution, injection of an intercalating agent, electrochemical measurement, storage of measurement data, and target base sequence based on the measurement data Up to the determination can be performed automatically. Thereby, the reproducibility and detection accuracy of the detection signal can be improved, and the time until the result is derived can be reduced.
[0214]
The present invention is not limited to the above embodiment.
[0215]
Although the case where the probe to be immobilized on the working electrode 501 is a DNA probe has been described, it may be a probe composed of a nucleic acid other than DNA, or a probe having a predetermined base sequence other than nucleic acid.
[0216]
The sharing of the processing between the computer 16 and the control mechanism 15 is not limited to the above. For example, if the measurement system 12, the liquid supply system 13, and the temperature control mechanism 14 have a processor that interprets a command from the computer 16 and executes each component, the control mechanism 15 may be omitted. In this case, the function of the control mechanism 15 as shown in FIG. 31 is executed by the computer 16.
[0219]
The management of the timing of the measurement system 12, the liquid supply system 13, and the temperature control mechanism 14 is performed by managing the processor if the measurement system 12, the liquid supply system 13, and the temperature control mechanism 14 have a processor for managing the timing. Each process is executed based on the timing of the execution. In this case, if the computer 16 transmits the automatic analysis condition parameters to the measurement system 12, the liquid supply system 13, and the temperature control mechanism 14, there is no need to manage the timing.
[0218]
Further, the computer 16 may perform timing control of the measurement system 12, the liquid feeding system 13, the temperature control mechanism 14, and the control mechanism 15.
[0219]
Further, the example in which the sample inlet 119 is communicated with the delivery port 116b has been described, but the sample inlet 119 may be communicated with the delivery port 116a. Further, the case where the number of the input port 116a and the number of the output port 116b is one each is shown, but the present invention is not limited to this. For example, the number of ports may be increased to any number such that one input port 116a is provided at the center of the cell 115 and plural output ports 116b are provided at a plurality of radial positions on the periphery of the cell 115. Furthermore, the example in which the inlet port 116a and the outlet port 116b extend perpendicularly to the cell bottom surface is shown, but the present invention is not limited to this, and is configured to extend parallel to the cell bottom surface. Is also good. Further, although the electrodes 21a and 21b and the bonding pads 221 on the base sequence detection chip 21 are shown in a laminated structure of Ti or Au, electrodes and pads using other materials may be used. Further, the arrangement of the electrodes 21a and 21b is not limited to that shown in FIG. The numbers of electrodes of the working electrode 501, the counter electrode 502, and the reference electrode 503 are not limited to those illustrated.
[0220]
Further, the liquid feeding system 13 is not limited to the one shown in FIG. For example, a more complicated reaction in the cell 115 can be performed by adding a supply system that supplies a chemical solution or gas other than air, Milli-Q water, a buffer, and an intercalating agent according to the type of reaction. The control of the supply path and supply amount of the chemical solution and air between the respective pipes may be performed by means other than the solenoid valve. The operation of the liquid feeding system 13 shown in FIG. 20 is only an example, and can be variously changed according to the purpose of the reaction and the like.
[0221]
Further, FIGS. 32 to 34 show the case where the automatic base sequence analyzer 1 is used for type determination, but this is only an example, and may be used for other analysis purposes. Also, the automation method shown in FIG. 35 is only an example, and the automation sequence is variously changed by variously changing the configurations of the chip cartridge 11, the measuring system 12, the liquid feeding system 13, the temperature control mechanism and the control mechanism 15. Is done.
[0222]
Although the working electrode 501, the counter electrode 502, and the reference electrode 503 are all arranged on the base sequence detection chip 21, the present invention is not limited to this. For example, only the working electrode 501 may be arranged on the base sequence detection chip 21, and the counter electrode 502 and the reference electrode 503 may be formed on the chip cartridge upper lid 112 side, or one of the counter electrode 502 and the reference electrode 503 may be used for base sequence detection. It may be arranged on the chip 21. Thus, the working electrode 501, the counter electrode 502, and the reference electrode 503 may be three-dimensionally arranged on different planes.
[0223]
Further, the relationship between the base sequence detection chip 21 and the chip cartridge upper lid 112 may be used upside down.
[0224]
【Example】
An example in which SNPs are detected using the base sequence detection device of the above-described embodiment will be described below. Here, the present invention is applied when discriminating whether the nucleotide sequence of the SNPs of the MxA-88 position gene is G / G type, T / T type, or G / T heterozygous.
[0225]
A DNA probe having a sequence complementary to the MxA gene is immobilized on the working electrode 501 of the base sequence detection chip in advance. Four types of probe DNA fragments in which the base at the SNP position is replaced with ATGC and a DNA fragment having a completely different sequence (called a negative control) are immobilized on separate electrodes (working electrode 501). Here, 200 nL of each probe modified with cysteine at the N-terminus was spotted and left for 1 hour to immobilize on the working electrode 501 made of Au. In this way, the printed circuit board 22 in which the prepared base sequence detection chip 21 is sealed is mounted on the chip cartridge 11.
[0226]
Next, a target DNA whose base at the SNP position is G-type is dissolved in a 2 × SSC-1 mmol / L EDTA solution, and then injected into the cell 115 from the sample injection port 119 using a pipette or the like. Here, the sample solution flows from the sample inlet 119 on the delivery port 116b side to the delivery port 116a side while filling the cell 115 with the solution. Since the inlet port 116a is formed at a position in contact with the sealant 24, the sample solution can be completely filled into the cell 115 without leaving air bubbles in the cell 115.
[0227]
Next, the chip cartridge 11 is mounted on the main body of the apparatus (measurement system 12, liquid supply system 13, temperature control mechanism 14), and the computer program is started by the computer 16, so that the subsequent processing is automatically performed. Is
[0228]
Here, the contents of the automatic processing will be described. First, a reaction (hybridization) is performed at 45 ° C. for 15 minutes. Then, the 0.2 × SSC-1 mmol / L EDTA solution is sent into the cell 115 by controlling the electromagnetic valve and the pump of the solution sending system 13. Then, while the cell 115 is filled with the solution, the solution is held at 55 ° C. for 30 minutes to wash the DNA non-specifically adsorbed on the electrodes 211 and 212 having different sequences on the base sequence detection chip 21. Next, a 10 μmol / L Hoechst 33258 solution is sent into the cell 115. Then, with the cell 115 filled, the measurement system 12 measures the oxidation current from the Hoechst 33258 at each working electrode 501.
[0229]
Next, the computer 16 extracts a region corresponding to the Hoechst oxidation current from the current / voltage characteristic curve by the analysis program, and derives a peak current value for each electrode (working electrode 501). Further, statistical processing such as type determination filtering is performed according to the algorithm of the analysis program, and the type of the target DNA is determined. The obtained judgment result is displayed on the display of the computer 16. As a result, the signal intensity from the electrode having the probe sequence corresponding to the C-type probe was the highest, and the base sequence at the SNP position of the target DNA was determined to be G-type.
[0230]
The uniformity of the current value for the same type of electrode within the surface of the base sequence detection chip 21 was within 3% in CV value. As a result, the reliability of SNPs detection was improved as compared with the conventional method.
[0231]
【The invention's effect】
As described in detail above, according to the present invention, in-plane uniformity in a cell can be improved.
[0232]
Further, according to another aspect of the present invention, it is possible to automatically execute the processing including the detection of the base sequence and the analysis of the detected data, so that the reproducibility of data and measurement is improved.
[Brief description of the drawings]
FIG. 1 is a conceptual diagram showing the entire configuration of a base sequence detection device according to one embodiment of the present invention.
FIG. 2 is a diagram showing details of a configuration of a chip cartridge according to the embodiment.
FIG. 3 is a view showing the support and the chip cartridge upper lid before being fixed with the upper lid fixing screw according to the embodiment.
FIG. 4 is a diagram showing a detailed configuration of a printed circuit board on which the base sequence detection chip according to the embodiment is mounted.
FIG. 5 is a view showing a cell and a chemical supply system leading to the cell according to the embodiment;
FIG. 6 is an exemplary view showing a modification of the cell according to the embodiment;
FIG. 7 is an exemplary view showing a more detailed configuration of a positional relationship between components near a cell according to the embodiment;
FIG. 8 is an exemplary view showing a modification of each component near the cell according to the embodiment;
FIG. 9 is a sectional view of a modification of the shape of the cell according to the embodiment;
FIG. 10 is an exemplary sectional view of a modification of the shape of the cell according to the embodiment;
FIG. 11 is an exemplary sectional view of a modification of the shape of the cell according to the embodiment;
FIG. 12 is a cross-sectional view of a modification of the positional relationship between the cell, the liquid supply port, and the liquid discharge port according to the same embodiment.
FIG. 13 is a cross-sectional view of a modification of the positional relationship between the cell and the liquid sending port and the liquid discharging port according to the same embodiment.
FIG. 14 is a sectional view of a modified example of the positional relationship between the cell and the liquid sending port and the liquid discharging port according to the same embodiment.
FIG. 15 is a sectional view of a modified example of the positional relationship between the cell and the liquid sending port and the liquid discharging port according to the embodiment.
FIG. 16 is a cross-sectional view of a modification of the positional relationship between the cell and the liquid supply port and the liquid discharge port according to the same embodiment.
FIG. 17 is a process sectional view of the method for manufacturing a base sequence detection chip and a printed circuit board according to the embodiment.
FIG. 18 is a top view of the base sequence detection chip according to the same embodiment.
FIG. 19 is a view showing an example of a specific configuration of a liquid feeding system according to the embodiment.
FIG. 20 is a view showing a flowchart of a liquid sending step for detecting a base sequence using the liquid sending system according to the embodiment.
FIG. 21 is a view showing a specific configuration of a measurement system according to the embodiment.
FIG. 22 is a diagram showing a configuration of a conventional potentiostat.
FIG. 23 is a view showing voltage characteristics according to the embodiment.
FIG. 24 is a diagram showing voltage characteristics of a conventional potentiostat.
FIG. 25 is a diagram showing a current / voltage characteristic curve applied to a counter electrode in the potentiostat according to the embodiment and a conventional potentiostat.
FIG. 26 is a view showing a modified example of the potentiostat according to the embodiment.
FIG. 27 is a view showing a modified example of the potentiostat according to the embodiment.
FIG. 28 is a view showing a modified example of the potentiostat according to the embodiment.
FIG. 29 is a view showing a modified example of the potentiostat according to the embodiment.
FIG. 30 is an exemplary conceptual diagram showing the relationship between the control mechanism and other components of the computer according to the embodiment;
FIG. 31 is a diagram showing an example of a detailed configuration of a control mechanism according to the embodiment.
FIG. 32 is an exemplary view showing an example of a measurement data analysis technique according to the embodiment.
FIG. 33 is a view showing a flowchart of a type determination filtering process according to the embodiment.
FIG. 34 is an exemplary view showing an example of a type determination process according to the embodiment.
FIG. 35 is a sequence diagram of an automatic base sequence analysis method using the base sequence detection device according to the embodiment.
FIG. 36 is a diagram showing an example of an optimal cell shape according to the embodiment.
[Explanation of symbols]
1 ... Base sequence detector
10 Measurement unit
11 ... Chip cartridge
12… Measurement system
12a, 12b, 12c, 12d, 12e ... potentiostat
13 ... Liquid feeding system
14. Temperature control mechanism
15 ... Control mechanism
16 ... Computer
17… Local bus
21 ... Base sequence detection chip
21a ... Electrode (working electrode, counter electrode)
21b ... electrode (reference electrode)
22 Printed circuit board
22a ... electrical connector
23 ... sealing resin
24, 24a ... sealing material
25 ... Board fixing screw
110 ... chip cartridge body
111 ... Support
112 ... Chip top cover
113a, 113b ... interface unit
114a, 114b ... flow path
115 ... cell
115a, 115b ... cell hole
115c, 115e, 115f, 115g, 115h ... straight line
115d ... counterbore hole
116a ... Discharge port
116b: Liquid supply port
117: Upper cover fixing screw
117a ... Screw hole
119 ... Sample inlet
120 ... lid
121 ... Seal material
500 ... Protection circuit
501 ... working electrode
502 ... opposite pole
503: Reference electrode
510 ... voltage pattern generation circuit

Claims (8)

A base sequence detection device that detects a base sequence in the sample by an electrochemical reaction between the sample and a probe having a predetermined base sequence in the flow path,
A substrate, a plurality of base sequence detection chips provided on the substrate and including a plurality of electrodes to which the probes are immobilized,
A cell is provided on the substrate to surround the plurality of electrodes, and a cell that seals a chemical solution or air on the substrate,
An inlet port that opens on the upper surface of the cell and that sends a chemical solution or air into the cell,
A delivery port that opens to the cell upper surface and that delivers a chemical solution or air from inside the cell,
A sample injection port provided to be open on the cell upper surface, and injecting a sample into the cell,
The width of the cell in a direction perpendicular to the flow path of the chemical or air from the inlet port to the outlet port formed in the cell is formed smaller than the distance between the inlet port and the outlet port. A base sequence detection device. A base sequence detection device that detects a base sequence in a sample by an electrochemical reaction of a sample and a probe having a predetermined base sequence in a cell defined by a cell top surface, a cell side surface, and a cell bottom surface,
A substrate, comprising a plurality of electrodes formed on the substrate and to which the probes are immobilized, a base sequence detection chip that defines the cell bottom surface by the substrate surface,
A seal material that is provided so as to surround the plurality of electrodes by a seal material inner periphery from a seal material bottom surface to be in contact with the substrate to a seal material upper surface having a predetermined height, and defines a cell side surface;
A chip cartridge upper lid that abuts on the upper surface of the sealing material to close the upper surface of the sealing material and define the upper surface of the cell;
An inlet port that opens on the upper surface of the cell of the chip cartridge upper lid, and that sends a chemical solution or air into the cell,
A delivery port that opens on the cell upper surface of the chip cartridge upper lid and that delivers a chemical solution or air from inside the cell,
A sample injection port for injecting a sample into the cell, provided on the chip cartridge upper lid,
The width of the cell in a direction perpendicular to the direction of the flow path of the chemical or air from the inlet port to the outlet port formed in the cell is greater than the distance between the inlet port and the outlet port. A base sequence detection device formed small. The peripheral portion of the opening of the inlet port to the cell upper surface is formed to be in contact with or overlap with the peripheral portion of the cell upper surface,
The base sequence detection device according to claim 2, wherein a peripheral portion of the opening of the delivery port on the upper surface of the cell is formed apart from a peripheral portion of the upper surface of the cell. The base sequence detection device according to claim 2, wherein the sample inlet is provided in communication with the delivery port. The base sequence detection device according to claim 1 or 2,
A first pipe that communicates with the inlet port and supplies a chemical or air into the cell through the inlet port, and a first valve that controls a flow rate of the chemical or air in the first pipe. And a second pipe that communicates with the delivery port and discharges a chemical or air from inside the cell through the delivery port, and controls a flow rate of the chemical or air in the second pipe. A second valve, a discharge system provided in the second pipe, and provided with a pump for sucking up a chemical solution or air from inside the cell;
A measurement system including a voltage application unit that applies a voltage between the plurality of electrodes and another counter electrode,
A temperature control mechanism for controlling the temperature of the base sequence detection chip,
Controlling a first valve of the supply system, a second valve and a pump of the discharge system, the voltage application unit of the measurement system, and the temperature control mechanism, and an electrochemical reaction signal from the plurality of electrodes. And a control mechanism for storing the electrochemical reaction signal as measurement data,
An automatic base sequence analysis apparatus comprising: a computer that controls the control mechanism by giving control condition parameters to the control mechanism and that executes a base sequence analysis process based on the measurement data. A voltage pattern generating circuit that generates a voltage pattern to be applied to the counter electrode;
A feedback circuit that feeds back a voltage of a reference electrode provided in the cell to the counter electrode, and an amplifier that amplifies a voltage applied to the counter electrode based on a feedback voltage of the voltage pattern generation circuit and the feedback circuit.
The automatic base sequence analysis apparatus according to claim 5, further comprising: a resistor provided between the amplifier or the amplifier and the counter electrode; and a protection circuit including a switch for performing on / off control of a current flowing through the resistor. . 6. The automatic base sequence analyzer according to claim 5, wherein the base sequence detector comprises a plurality of units, and the control mechanism is provided one for each of the base sequence detectors. The computer is
A plurality of first electrochemical signals obtained from a plurality of first probes having a first target complementary base sequence complementary to the first target base sequence having the base at the SNP position as a first base; Among the second electrochemical signals obtained from the plurality of second probes having the second target complementary base sequence complementary to the second target base sequence in which the base at the position is the second base, the signal value is The larger one is selected, the plurality of first electrochemical signals and the plurality of second electrochemical signals are tested, and if it is determined that there is a significant difference from each other, the base at the SNP position in the sample is When it is determined that the first base or the second base is homozygous and it is determined that there is no significant difference, the base at the SNP position in the sample is a heterotype of the first base and the second base. Comprising a type determination processing unit for performing a type determination process for determining that Nucleotide sequence automatic analyzer according to 5.

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