JP4417946B2 - Automatic nucleotide sequence analyzer - Google Patents

Description

  The present invention relates to a base sequence detection device that detects a base sequence, and an automatic base sequence analysis device that automatically controls the base sequence detection device and automatically analyzes a measurement signal.

  Conventionally, for example, there are devices that perform only hybridization, devices that perform only electrochemical measurements after adding an intercalating agent after this hybridization, or devices that automatically perform from washing to washing with a buffer. Was.

  When the measurement is performed using the apparatus as described above, when each process is completed, the operator needs to manually transfer the sample to the apparatus for the next process, and is thus restricted in time. In addition, since an operator is involved in the transfer between processes, there is a problem that the reproducibility of data between samples is poor.

  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 caused by, for example, in-plane non-uniformity of reaction in a cell in which an electrochemical reaction is performed.

  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 apparatus that improves in-plane uniformity in a cell.

  Another object of the present invention is to provide an automatic base sequence analyzing apparatus capable of performing automatically from reaction to liquid feeding and measurement.

  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 in a flow path and a probe having a predetermined base sequence, the substrate and the substrate A base sequence detection chip provided with a plurality of electrodes and a plurality of electrodes on which the probe is immobilized, and 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 that opens to the top surface of the cell and feeds chemical or air into the cell, a delivery port that opens to the top surface of the cell and sends the chemical or air from within the cell, and the cell And a sample injection port for injecting a sample into the cell, the chemical solution or air flow from the inlet port to the outlet port formed in the cell. 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.

  According to another aspect of the present invention, a base for detecting a base sequence in a sample by an electrochemical reaction between the sample in the cell defined by the cell upper surface, the cell side surface and the cell bottom and a probe having a predetermined base sequence A sequence detection device comprising a substrate and a plurality of electrodes formed on the substrate to which the probe is immobilized, a base sequence detection chip for defining the cell bottom surface by the substrate surface, and a contact with the substrate The seal material is provided from the bottom surface of the seal material in contact with the top surface of the seal material having a predetermined height so as to surround the plurality of electrodes by the inner periphery of the seal material, and is in contact with the seal material that defines the cell side surface and the top surface of the seal material A top surface of the chip cartridge that covers the top surface of the sealing material and defines the top surface of the cell; an opening on the top surface of the cell of the top lid of the chip cartridge; An infeed port for injecting air; an opening port on the top surface of the cell of the chip cartridge upper lid; a delivery port for delivering a chemical solution or air from within the cell; and a top lid of the chip cartridge for providing a sample in the cell. A sample injection port for injection, and the width of the cell in a direction perpendicular to the direction of the flow path of the chemical solution or air from the inlet port to the outlet port formed in the cell, There is provided a base sequence detection device formed smaller than the distance between the sending port and the sending port.

  According to still another aspect of the present invention, there is provided a base sequence detection device as described above, a first communicating with the infeed port, and supplying a chemical solution or air into the cell through the infeed port. And a supply system comprising a first valve for controlling the flow rate of the chemical or air in the first pipe, and the chemical solution or air from the inside of the cell through the delivery port. And a second valve for controlling the flow rate of the chemical liquid or air in the second pipe, and a pump provided in the second pipe for sucking up the chemical liquid or air from the cell. A discharge system; 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 that controls the temperature of the base sequence detection chip; and the liquid supply system The first valve, the second valve and the pump, and the electric power of the measurement system A control mechanism for controlling the application unit and the temperature control mechanism, detecting an electrochemical reaction signal from the plurality of electrodes, and storing the electrochemical reaction signal as measurement data, and giving a control condition parameter to the control mechanism Thus, there is provided an automatic base sequence analyzing apparatus comprising a computer for controlling the control mechanism and executing base sequence analysis processing based on the measurement data.

  Further, the present invention relating to an apparatus is also established as an invention of a method realized by the apparatus.

  Further, the present invention relating to an apparatus or a method is also 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.

  According to the present invention, in-plane uniformity within a cell can be improved.

  Further, according to another aspect of the present invention, since it can be automatically executed including the detection of the base sequence and the analysis of the detected data, the reproducibility of data and measurement is improved.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a conceptual diagram showing the overall configuration of a base sequence automatic analyzer according to an embodiment of the present invention. As shown in FIG. 1, a base sequence automatic analyzer 1 includes a chip cartridge 11 (base sequence detector), a measurement system 12 electrically connected to the chip cartridge 11, and a channel provided in the chip cartridge 11. And a temperature control mechanism 14 that controls the temperature of the liquid supply system 13 and the chip cartridge 11 that are physically connected via the interface unit.

  These measurement system 12, liquid feeding system 13, and temperature control mechanism 14 are controlled by a control mechanism 15. The control mechanism 15 is electrically connected to the 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 feeding system 13, and the temperature control mechanism 14 are referred to as a measurement unit 10.

  A printed circuit board 22 on which a base sequence detection chip 21 on which a DNA probe is immobilized is mounted is used for the chip cartridge 11.

  In the following embodiments, the base sequence of DNA to be detected is referred to as a target base sequence. The target base sequence is complementary, and the base sequence that selectively reacts with the target base sequence is called a target complementary salt sequence. A DNA probe containing this target complementary base sequence is immobilized on the working electrode of the base sequence detection chip 21. The sample (specimen solution) introduced into the cell of the base sequence detection chip 21 contains DNA to be examined. The base sequence of the DNA to be tested is referred to as the sample base sequence.

  In the base sequence detection apparatus of this embodiment, the target base sequence is contained in the sample by hybridizing the sample base sequence and the target complementary base sequence, and monitoring the presence or absence of the reaction after introducing the buffer and the intercalating agent. It is determined whether or not.

  2A and 2B are diagrams showing a detailed configuration of the chip cartridge 11, wherein FIG. 2A is a view seen from the top, FIG. 2B is a view seen from the AA direction, and FIG. 2C is a view seen from the BB direction. FIG. 4D is a partial perspective cross-sectional view, and FIG. 5D is a view of the support 111, which is one component of the chip cartridge 11, viewed from the back surface.

  The chip cartridge main body 110 includes a support 111 that supports the printed circuit board 22 from the lower side, and a chip cartridge upper lid 112 that sandwiches and fixes the printed circuit board 22 from the upper side together with the support 111.

  Two openings are provided in the side portion of the chip cartridge upper lid 112, and an interface portion 113a is connected to one of the openings, and an interface portion 113b is connected to the other one. These interface units 113 a and 113 b function as an interface between the liquid feeding system 13 and the chip cartridge 11.

  Channels 114a and 114b are provided in the interface portions 113a and 113b, respectively. The chemical solution and air from the upstream side of the liquid feeding system 13 are fed into the chip cartridge 11 through 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 feeding system 13 through the flow path 114b.

  In FIGS. 2A to 2C, the channels 114a and 114b are indicated by broken lines. These flow paths 114a and 114b communicate from the interface sections 113a and 113b to the inside of the chip cartridge upper lid 112, and further communicate with the cell 115 indicated by a circular broken line in FIG. 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. This cell 115 is sealed with the base sequence detection chip 21 when the four corners of the printed circuit 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 substrate fixing screw 25. It is defined by a closed space region surrounded by the material 24 and the chip cartridge upper lid 112. In a state where the printed circuit board 22 on which the base sequence detection chip 21 is mounted is fixed to the chip cartridge upper cover 112, the printed circuit board 22 is held by the support 111 and the chip cartridge upper cover 112 with the sealing material 24 interposed therebetween. Further, the chip cartridge upper lid 112 is fixed by the upper lid fixing screw 117. As a result, various chemical and air injection / discharge paths communicating 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 the printed board 22 with a sealing resin 23.

  The chip cartridge upper lid 112 located on the upper surface of the cell 115 is provided with an infeed port 116a and an outfeed port 116b. The inlet port 116a penetrates from the side surface to the bottom surface of the chip cartridge upper lid 112, and opens to the bottom surface of the chip cartridge upper lid 112 at the cell hole portion 115a. The delivery port 116b penetrates from another side surface to the bottom surface of the chip cartridge upper lid 112, and opens to the bottom surface of the chip cartridge upper lid 112 through the cell hole portion 115b. By connecting the inflow port 116a to the flow path 114a and the output port 116b to the flow path 114b, the flow path 114a and the cell 115, and the flow path 114b and the cell 115 communicate with each other.

  An electrical connector 22 a is set on the surface of the printed circuit board 22 and at a position separated from the cell 115. The electrical connector 22 a is electrically connected to the lead frame of the board body of the printed board 22. In addition, the lead frame of the substrate body is electrically connected to various electrodes of the base sequence detection chip 21 by leads or the like. By connecting the terminal of the measurement system 12 to the electrical connector 22a, an electrical signal obtained by the base sequence detection chip 21 is further transmitted through a predetermined terminal provided at a predetermined position of the printed circuit board 22, and further the electrical connector. It can output to the measurement system 12 via 22a.

  As shown in FIG.2 (d), the support body 111 is U-shaped, and the notch part 111a is provided in the center. The notch 111a is smaller than the printed board 22 and larger than the base sequence detection chip 21. Thereby, 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. 117a is a screw hole to which the upper lid fixing screw 117 is fixed.

  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 DNA reaction is generally performed in a temperature range relatively close to room temperature. Therefore, the temperature control using only the heater is poor in stability. In addition, since it is necessary to control the DNA reaction according to the temperature profile, a separate cooling mechanism is required. In that respect, the Peltier element is optimal because it can be heated or cooled by changing the direction of the current.

  FIG. 3 is a view 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, the four corners of the printed circuit board 22 on which the base sequence detection chip 21 is mounted are fixed to the chip cartridge upper lid 112 by the board fixing screws 25. A sealing material 24 is integrated with the chip cartridge upper lid 112. Therefore, the cell 115 surrounded by the sealing material 24 and the chip cartridge upper lid 112 is defined on the base sequence detection chip 21. Further, the chip cartridge upper lid 112 is fixed to the support 111 with the upper lid fixing screw 117 and used. The board fixing screw 25 may be fixed from the back surface side of the printed circuit board 22 or from the front surface side. In this way, by fixing the printed circuit board 22 to the chip cartridge upper lid 112, the adhesion between the base sequence detection chip 21, the sealing material 24, and the chip cartridge upper lid 112 can be reliably maintained.

  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 with a sealing resin 23. On the base sequence detection chip 21, for example, an electrode 21a that operates as a working electrode or a counter electrode and an electrode 21b that operates as a reference electrode are provided. In FIG. 4, the case where one electrode 21a is provided in each of the four regions divided by the electrode 21b is schematically shown. However, one or a plurality of 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. For reference, the area where the cell 115 is defined is shown by a broken line in FIG. As shown in FIG. 4, the cell 115 is determined so that the electrodes 21 a and 21 b and the like are located in the region of the cell 115.

  An electrical connector 22 a is installed at the 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 or the like provided on the surface of the printed board 22. By connecting the signal interface of the measurement system 12 to the electrical connector 22a, each electrode of the base sequence detection chip 21 and the measurement system 12 can be electrically connected.

  FIG. 5A is a cross-sectional view of the cell 115 shown in FIG. 2A and the chemical solution supply system leading to the cell 115 as seen from the CC direction, and FIG. 5B is a top view of the vicinity of the cell 115.

  As shown in FIG. 5A, for example, the sealing material 24 such as a packing ring is a cell 115 in a closed space formed by being tightly fixed by the chip cartridge upper lid 112 and the base sequence detection chip 21. The bottom surface of the cell 115 is defined 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 a gap, and the upper surface of the sealing material 24 is in contact with the lower surface of the chip cartridge upper lid 112 without a gap. Thus, a completely closed space is defined except for the cell hole portions 115a and 115b, and liquid tightness between the base sequence detection chip 21 and the lid 120 is maintained.

  The cross-sectional shape of the cell 115 is circular as shown in FIG. An inlet port 116a and an outlet port 116b having a circular cross section are provided on the outer edge of the circular cell 115 at substantially symmetrical positions with the center of the circle of the cell 115 in between. The outer peripheries of the sending port 116 a and the sending port 116 b are substantially in contact with the outer periphery of the cell 115. In addition, the central axes of the circular cross sections of the inlet port 116 a and the outlet port 116 b are located on a straight line 115 c passing through the center of the circular bottom surface of the cell 115.

  The sending port 116a and the sending 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 inflow port 116a and the outflow port 116b are further bent in the direction away from the center of the cell 115, and are connected to the channels 114a and 114b, respectively.

  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 bent at a substantially right angle in a direction away from the center of the cell 115, but 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 injection port 119. Thus, the sample injected from the sample injection port 119 is introduced into the cell 115 through the delivery port 116b. The central axes of the sample injection port 119 and the delivery port 116b are substantially coincident, and the diameter of the sample injection port 119 is set larger than the diameter of the delivery port 116b. Further, the sample injection port 119 can be closed by the lid 120 provided in the vicinity of the sample injection port 119. Accordingly, when the chemical liquid is circulated from the flow path 114a to the flow path 114b through the cell 115 without using the sample injection port 119, the chemical liquid can be prevented from flowing out of the sample injection port 119. A route can be secured. Further, the lid 120 is provided with a sealing material 121. By sealing the sample injection port 119, slight leakage of the chemical solution can be prevented. Although not particularly shown in the example of FIG. 5 (a), a sealing material 121 having such a depth as to completely block the path to the sample inlet 119, leaving only the path connected from the delivery port 116b to the flow path 114b. If used, it is possible to reduce stagnation of chemicals and air to the sample inlet 119 side.

  With the above-described configuration, the chemical solution and air can flow in the order indicated by the arrows in FIG. 5A in the order of the flow path 114a, the infeed port 116a, the cell 115, the outfeed port 116b, and the flow path 114b. A sample is injected from the sample inlet 119 and introduced into the cell 115 through the delivery port 116b in the direction of the arrow. Therefore, the sample is injected from the delivery side, and the flow of the chemical solution supply and the sample injection path are set in reverse. Thereby, the cleaning efficiency of the sample can be increased in the cleaning step.

FIG. 6A is a modification of the portion surrounded by the broken line in FIG. 5A, and FIG. 6B is a view of the cell 115 in the modification of FIG. As shown in FIG. 6A, the inlet port 116a has a counterbore hole 115d. That is, the diameter of the inlet port 116a gradually increases toward the counterbore hole 115d, and the diameter of the position away 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. Spot facing hole 115d has a larger diameter _{d 11} than the diameter _{d 1} of the inlet port 116a. The outer periphery of the infeed port 116a and the inner periphery of the sealing material 24 are arranged substantially coincident with each other. Therefore, a part of the outer periphery of the counterbore hole 115 d is positioned so as to overlap with 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 elliptical 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 thereto. But you can.

  Although FIG. 6 shows the case where the counterbored hole 115d is provided in the inlet port 116a, a similar counterbored hole may be provided in the outlet port 116b.

  Thus, by providing the counterbore hole 115d in the opening of the cell 115, the introduction port to the cell 115 has a funnel shape, and it is easy to suck out the chemical liquid and bubbles, and the chemical liquid and bubbles are unlikely to remain in the cell 115. Has an effect.

FIG. 7 is a diagram showing a more detailed configuration of the positional relationship of each component in the vicinity of the cell 115, FIG. 7 (a) is a cross-sectional view, and FIG. 7 (b) is a top view. In FIG. 7A, d ₄ is the height of the sealing material 24. In FIG. 7 (b), _{d 0} is the distance from the central axis O of the cell 115 to the periphery, _{d 3} is the outer diameter of the sealing member _{24, d 1,} d ₂ is the port cross-section of inlet ports 116a and outlet port 116b feed respectively D ₁ <d ₃ , d ₂ <d ₃ . Assuming that the central axes of the inlet port 116a and the outlet port 116b are O ₁ and O ₂ , the distance between the axis O and the axis O ₁ is d ₀₁ , and the distance between the axis O and the axis O ₂ is d ₀₂ , d ₀₁ + d _{1 = d 0, d 02 + d} 2 = d 0 is established. The radius d _{0 of the} cell 115 is set to about 3 mm to 30 mm, and the radii d ₂ and d ₃ of the sending port 116a and the sending port 116b are set to about 0.3 mm to ₂ mm, respectively. The height _{d 4} of the sealing member 24 is set to about 0.5 mm.

  In this way, by opening the inlet port 116a and the outlet port 116b at the periphery of the upper surface of the cell 115 having a circular bottom surface, the chemical solution is transferred to the cell when the inside of the cell 115 is replaced with air when the chemical solution is supplied. It is possible to prevent the remaining chemical solution from coming out of the inside 115. In addition, even when a chemical solution is introduced into the cell 115 filled with air, the cell 115 can be filled without remaining air. In addition, since the sample injection port 119 is also provided so as to communicate with the delivery port 116b, air remaining in the cell 115 during sample injection can be prevented, and the cell 115 can be filled with the sample.

  Thereby, the in-plane uniformity in the cell 115 of the current characteristic obtained in the cell 115 by the electrochemical reaction of the DNA probe is improved, and the reliability of SNP detection is improved.

  FIG. 7C shows a state in which the sealing material 24 is fixed tightly between the chip cartridge upper lid 112 and the base sequence detection chip 21 with 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 sealing material 24 is placed on the base sequence detection chip 21, the chip cartridge upper lid 112 is fixed with the substrate fixing screw 25, thereby providing a gap between the upper surface of the sealing material 24 and the chip cartridge upper lid 112. The contact is made without any gap, and the lower surface of the sealing material 24 and the surface of the base sequence detection chip 21 are brought into contact with no gap, so that the cell 115 is defined.

  Note that receiving holes may be provided outside the cell hole portions 115a and 115b of the chip cartridge upper lid 112 in order to position 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 sealing material 24. . As a result, the sealing material 24 is fitted into the receiving hole, and the sealing material 24 is positioned with respect to the chip cartridge upper lid 112.

FIG. 8 is a diagram showing a modification of each component in the vicinity of the cell 115 shown in FIG. FIG. 8A is a cross-sectional view, and FIG. 8B is a top view. Components common to those in FIG. 7 are denoted by the same reference numerals, and detailed description thereof is omitted. FIG. 8 differs from FIG. 7 in that the chip cartridge upper lid 112 and the seal material 24a having a height d ₄₁ are used, and the chip cartridge upper lid 112 has substantially the same inner diameter and outer diameter as the seal material 24a. This is to provide a ring-shaped convex portion 112 a having a height d ₄₂ provided on the bottom surface of the chip cartridge upper lid 112. 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 of d ₀ and an outer diameter of d ₃ . The ring-shaped convex portion 112a is preliminarily printed with a sealing material 24a by, for example, screen printing, and is formed integrally with the sealing material 24a. Accordingly, the cell 115 can be determined without positioning the sealing material and the chip cartridge upper cover as in the configuration shown in FIG. 7, and the assembly process of the cell 115 is simplified.

As shown in FIG. 8C, the chip cartridge upper lid 112 having the sealing material 24a fixed in advance is pressed and fixed to the base sequence detection chip 21 in the direction of the arrow using the upper lid fixing screw 117. The cell 115 can be easily defined. Further, by designing the heights d ₄₁ and d ₄₂ to be d ₄ = d ₄₁ + d ₄₂ with respect to the height d ₄ of the sealing material in FIG. 7, the cells 115 having the same height as in FIG. Can be determined. Further, the sealing material 24a and the ring-shaped convex portion 112a may be integrated using a method other than screen printing. For example, the integration may be realized by providing a projection on one of the sealing material 24a or the ring-shaped convex portion 112a and providing a hole that fits the projection on the other. In this case, there is an advantage that the sealing material 24a can be freely detached from the chip cartridge upper lid 112.

  Cross-sectional views of modifications of the shape of the cell 115 are shown in FIGS.

As shown in FIG. 9A, the cross section (bottom surface) of the cell 115 has a circular shape with a radius d ₀ , and a cylindrical sealing material 24 with an outer diameter d ₃ is disposed so as to surround the cell 115. The configuration shown in FIG. 9A is common to the configuration shown in FIGS.

In FIG. 9B, the shape of the cell 115 is a polygon. Further, the sealing material 24 is common in that it has an outer periphery with an outer diameter d ₃ , but the inner periphery 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 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, since the chemical | medical solution residue and the air residue in the position largely distant from the straight line 115c which connected the sending port 116a and the sending port 116b decrease, the in-plane uniformity of various electrochemical reactions for a measurement improves. In FIG. 9B, an octagonal shape is shown as one form of the polygon. However, the present invention is not limited to this, and it is needless to say that a polygon such as a triangle, a quadrangle, a pentagon, and the like may be used.

In FIG. 10A, the shape of the cell 115 is an ellipse. The outer periphery of the sealing member 24 is a circular shape of the outer diameter d _3, the inner periphery forms an elliptical shape. The center of the ellipse is O. The major axis of the ellipse is located on the line 115c, the length of the major axis is defined by 2 × d _0. Minor axis of the ellipse is located on a straight line 115e which is a straight line 115c through a vertical and and the center axis O, the length of the minor axis is defined by _{2 × d 5, d 5 <} d 0 is established. As described above, when the elliptical cell 115 is used, the width of the path of the chemical solution from the inlet port 116a to the outlet port 116b becomes smaller than that of the circular shape as shown in FIG. It becomes an elongated shape along. Accordingly, the chemical solution residue and the air residue at the position where the chemical solution path spreads are reduced, so that the in-plane uniformity of various electrochemical reactions for measurement is improved.

In FIG. 10B, the vicinity of the outer peripheral port of the cell 115 cross section is defined by a curve expressed by a multi-order equation. The outer periphery of the sealing material 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. Distance from the center O of the cell 115, to the intersection of the outer periphery and the line 115c of the cell 115 is _{d 0.} More specifically, when the intersection of the outer periphery of the cell 115 and the straight line 115c is the origin, the straight line 115c intersects 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 a predetermined multi-order equation. The equation can be set freely, and can be set, for example, to the shape of a parabola by setting.

  Note that this curve defines only the outer periphery of the half of the cell 115 on the side of the input port 116a, and the cell outer peripheral shape of the remaining half of the output port 116b by the curve of the same equation obtained by defining the x axis as the straight line 115g. Is determined. As a result, the outer peripheral shape of the cell 115 on the upstream side where the input port 116a is provided and the downstream side of the cell 115 where the output port 116b is provided forms a line-symmetric shape with the straight line 115e as a boundary.

  In the example of FIG. 10B, the cross-sectional shape of the cell 115 in the vicinity of the input port 116a is a parabola that extends toward the output port 116b. In addition, the cross-sectional shape of the cell 115 in the vicinity of the delivery port 116b is a parabolic shape spreading toward the delivery port 116a. As a result, when the region 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 region of the port corner has a circular cross section as shown in FIG. It becomes narrower than As a result, it is difficult for air and chemicals to remain in the port corner area, and the current characteristics of the measurement are improved. In addition, the width of the cell 115, that is, the width in the direction perpendicular to the flow path is narrower than the distance between the inlet port 116a and the outlet port 116b, as shown in FIGS. 9B and 10A. Is the same.

  FIG. 11A is a modification of FIG. In the case of FIG. 10B, the case where the outer peripheral shape of the cell 115 on the upstream side where the input port 116a is provided and the downstream side cell 115 where the output port 116b is provided is symmetrical with respect to each other is shown in FIG. ) Form an asymmetrical shape. If the intersection of the outer periphery of the cell 115 and the straight line 115c is the origin, the straight line 115c intersects the straight line 115c perpendicularly and the straight line passing through the origin is the 115f, the shape of the outer periphery of the cell 115 is a predetermined shape. It matches the shape of the curve of the multi-order equation.

  In the case of the figure, the equation is set to a different value on the input port 116a side and the output port 116b side. As a result, as shown in the figure, cells having an asymmetric shape are formed on the upstream side and the downstream side of the chemical liquid with the straight line 115e as a boundary. The sending port 116b side has a convex shape on the outside of the cell 115 and the sending port 116a side has a slightly convex shape on the outside of the cell 115, but its curvature is larger than that on the sending port 116b side.

  Needless to say, the shape of the cell 115 may be reversed on the input port 116a side and the output port 116b side from the case shown in FIG. Further, as will be described later, a slight gap may be provided between the position where the delivery port 116 b is formed and the position of the sealing material 24.

  Either the upstream or downstream cell periphery 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. It may be in the shape of a curve that cannot be performed.

  FIG.11 (b) is a modification of FIG.9 (b). Similarly to 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 a straight line 115c. The cell hole portions 115a and 115b are arranged so as to contact the outer periphery of the regular hexagon near the two apexes. 9B is the same as FIG. 9B in that the width of the cell 115, that is, the width in the direction perpendicular to the flow path is narrower than the distance between the input port 116a and the output port 116b.

  In FIGS. 9 (a), 9 (b), 10 (a), 10 (b), 11 (a), and 11 (b), the outer peripheral positions of the input port 116a and the output 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 has been described as an example. However, when the outer periphery of the input port 116a or the output port 116b and the cell 115 is within the cell 115, The present invention can also be applied to the case of protruding out of 115 (for example, when a counterbore hole 115d is used).

  Cross-sectional views of modifications of the positional relationship between the cell 115 and the input / output port 116a and the output port 116b are shown in FIGS. Note that the positions of the inlet port 116a and the outlet port 116b in FIGS. 12 to 16 indicate the outer peripheral positions of the cell hole portions 115a and 115b of these ports, that is, the opening positions with respect to the cells 115. When used, the outer peripheral position of the cross section of the counterbore hole 115d is shown.

  The positional relationship is different between the sending side / outgoing side, the case where the outer periphery of the cross section of the cell 115 and the sending port 116a or the sending port 116b overlap / contact / separate, the sending port 116a and the sending port 116b. There are at least 18 combinations depending on whether or not is at a symmetric position with respect to the central axis O of the cell 115 or at an asymmetric position.

  In addition, in FIGS. 12 to 16, as in the case shown in FIGS. 5, 7, and 9 (a), the case where the cross-sectional shape of the cell 115 is a circular shape will be described as an example. Also in the cases shown in FIGS. 10A, 10B, 11A, and 11B, the present invention can be similarly applied only by changing the cell shape.

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 seal material 24, and d ₁ and d ₂ are the inlet port 116a and the outlet port, respectively. radius 116b of the port section (cell pore part 115a and 115b cross section), send the central axis of the inlet port 116a and outlet port 116b _{O 1} and _{O 2} and then distance _{d 01} in the axial O and the axis _{O 1,} the axis O and the axis If the distance of O ₂ is d ₀₂ , d ₀₁ + d ₁ > d ₀ and d ₀₂ + d ₂ > d ₀ are established.

FIG. 12B shows a case where the outer periphery of the port overlaps the outer periphery of the cell 115 cross section 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 and d ₀₂ + d ₂ = d ₀ are established.

FIG. 13A shows the case where the outer periphery of the port is in contact with the outer periphery of the cell 115 cross section on the infeed side, and the outer periphery of the port overlaps the outer periphery of the cross section of the cell 115 on the sending side. The case where the configuration on the transmission side and the configuration on the transmission side are reversed is shown. In this example, d ₀₁ + d ₁ = d ₀ and d ₀₂ + d ₂ > d ₀ are established.

FIG. 13B shows a case where the outer periphery of the cross section of the cell 115 and the outer periphery of the port overlap on the sending side, and the outer periphery of the cross section of the cell 115 and the outer periphery of the port are separated on the sending side. In this example, d ₀₁ + d ₁ > d ₀ and d ₀₂ + d ₂ <d ₀ are established.

FIG. 14A shows a case where the outer periphery of the cell 115 cross section 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 and the outer periphery of the port overlap on the sending side. In this example, d ₀₁ + d ₁ <d ₀ and d ₀₂ + d ₂ > d ₀ hold.

FIG. 14B shows a case where the outer periphery of the cell 115 cross section and the outer periphery of the port are in contact with the sending side, and the outer periphery of the port is in contact with the outer periphery of the cross section of the cell 115 on the sending side. In this example, d ₀₁ + d ₁ = d ₀ and d ₀₂ + d ₂ = d ₀ are established. This example is common to the case shown in FIG.

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 and d ₀₂ + d ₂ <d ₀ are established.

FIG. 15B shows a case where the outer periphery of the cell 115 cross section 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 in contact with the outer periphery of the port on the sending side. In this example, d ₀₁ + d ₁ <d ₀ and d ₀₂ + d ₂ = d ₀ are established.

FIG. 16A 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 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 and d ₀₂ + d ₂ <d ₀ hold.

  As described above, FIGS. 12 to 15 and FIG. 16A show nine modifications according to the presence or absence of overlapping of ports and cells. FIG. 16B can be applied in combination with nine modified examples according to the presence or absence of overlapping of these ports and cells. In these nine modifications, 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 is shown, but FIG. 16B shows the case where the input port 116a and the output port 116b are located. 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 FIG. 16B with nine modified examples.

As shown in FIG. 16 (b), the straight line connecting the input port 116 a and the output port 116 b does not pass through the central axis O of the cell 115. That is, the straight line connecting the center O of the center _{O 1} and the cell 115 of the inlet port 116a 115 h does not match the straight line 115c connecting the center O of the center _{O 1} and the cell 115 of the delivery port 116 b. Therefore, the input port 116 a and the output port 116 b are provided at asymmetric positions with respect to the center O of the cell 115.

In FIG. 16 (b), the liquid feeding side overlap port periphery to the outer periphery of the cell 115 section, if吐液side port periphery is in contact with the outer periphery of the cell 115 section, ie _{d 01 + d 1> d 0} , d 02 + d _{Although 2} = d ₀ is satisfied (corresponding to FIG. 12B), the remaining eight modifications shown in FIGS. 12 to 15 and FIG. 16A excluding FIG. 12B are shown. Of course, the positional relationship may be applied.

  Of the combinations of the shapes of the cells 115 shown in FIGS. 9 to 16, the shapes considered to be optimal are shown in FIGS. 36 (a) and 36 (b). FIG. 36A shows an example in which the cell shape of FIG. 11A and the positional relationship of the ports of FIG. 13A are combined. FIG. 36B shows the cell shape of FIG. It is the example which combined the positional relationship of the port of a).

  As shown in FIGS. 36 (a) and 36 (b), when considering the flow path from the input port 116a to the output port 116b, the elongated cell shape in which the width of the flow path is narrower than the distance between the ports. It has become. Thereby, the chemical solution residue and the air residue at a point far away from the straight line connecting the delivery port 116a and the delivery port 116b are reduced, and the in-plane uniformity of the electrochemical reaction for measurement is improved.

  Further, the area of the port corner of the infeed port 116a is narrow, and the curvature of the curve that defines the cell periphery near the port corner is larger than the curvature of the curve that defines the cell periphery near the port corner of the output port 116b. Further, the peripheral edge of the inlet port 116a is formed so as to be in contact with or overlap with the peripheral edge of the upper surface of the cell 115. Further, the peripheral edge of the delivery port 116b is formed away from the peripheral edge of the upper surface of the cell 115. As a result, it is possible to reduce the remaining chemical liquid and air remaining easily in the vicinity of the port corner of the infeed port 116a during the feeding of the chemical liquid and air, and the feed generated at the port corner of the delivery port 116b during the feeding of the chemical liquid and air. Variations in liquid speed can be reduced, and air remaining can be reduced.

  Next, the manufacturing method of the base sequence detection chip 21 and the printed circuit board 22 described above will be described with reference to the process cross-sectional view of FIG.

  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.

Next, a Ti film 213 is formed on the entire surface of the substrate by sputtering, for example, with a thickness of 50 nm, and then an Au film 214 is formed, for example, with a thickness of 200 nm. 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 regions that will later become electrodes and wiring (FIG. 17A), and the Au film 214 and the Ti film 213 are etched (FIG. 17B). In this embodiment, a KI / I ₂ mixed solution was used for etching the Au film 214, and a NH ₄ OH / H ₂ O ₂ mixed solution was used for etching Ti. Etching of the Au film 214 includes a method using diluted aqua regia and a method of removing by ion milling. Similarly, the etching of the Ti film 213 can be performed by wet etching using hydrofluoric acid or buffered hydrofluoric acid, or by dry etching using plasma with a CF ₄ / O ₂ mixed gas, for example. is there.

  Next, the photoresist film 210 is removed by oxygen ashing (FIG. 17C). The removal process of the photoresist film 210 can be performed by using a solvent, a resist stripper, or a combination of these and an oxygen ashing process.

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. The hard baking method uses a hot plate, and the processing conditions can be changed as appropriate. Here, the photoresist film 215 is selected as the protective film, but it is also possible to use an organic film such as polyimide or BCB (benzocyclobutene) in addition to the photoresist. Further, an inorganic film such as SiO, SiO ₂ or SiN may be used for the protective film. In that case, the photoresist film 215 is opened so as to protect the electrode portion, and 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 over the entire surface, and then the electrode Alternatively, the photoresist film 215 may be patterned so as to open only the portion, the SiN film or the like on the electrode is removed by etching, and finally the photoresist film 215 is peeled off.

Next, dicing is performed to form a chip. Finally, in order to clean the surface of the electrode part, a treatment with a CF ₄ / O ₂ 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 electrical connector 22a is mounted. And the bonding pad of the base sequence detection chip 21 and the lead wiring on the printed circuit board 22 are connected by wire bonding. Thereafter, the wire bonding portion is protected using the sealing resin 23.

  Through the above steps, the printed circuit board 22 on which the base sequence detection chip 21 is mounted can be manufactured.

  A top view of the prepared base sequence detection chip 21 is shown in FIG. As shown in FIG. 18, a plurality of electrodes 21a are provided near the center of the chip surface. The rectangular 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 almost vertically so as to divide the set of the working electrode and the counter electrode into four substantially. 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 a broken line. A bonding pad 221 is disposed on the periphery of the chip. Each of the electrodes 21 a and the electrode 21 b is 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.

  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 flow path 114a side of the cassette 11 and a discharge system provided on the flow path 114b side.

An air supply source 401 is connected to the uppermost stream of the pipe 404. A check valve 402 is provided on the downstream side of the air supply source 401 to prevent a chemical solution other than air from flowing back to the air supply source 401 via the pipe 404, and further on the downstream side is a two-way electromagnetic valve. 403 (V _a ) is provided. As a result, the flow rate of air flowing from the pipe 404 toward the chip cartridge 11 is controlled.

The pipe 414 is connected to a milli-Q water supply source 411 that contains milli-Q water as one of the chemical solutions. A check valve 412 is provided on the downstream side of the milli-Q water supply source 411 to prevent a chemical solution or air other than the milli-Q water from flowing back to the milli-Q water supply source 411. A direction solenoid valve 413 ( _Vwa ) is provided. The three-way solenoid valve 413 switches communication between the pipe 404 and the pipe 415 and communication between the pipe 414 and the pipe 415. 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, supply of air and milli-Q water to the pipe 415 can be switched.

A buffer supply source 421 that contains a buffer (buffer solution) as one of the chemical solutions is connected to the pipe 424. On the downstream side of the buffer supply source 421, a check valve 422 for preventing a chemical solution or air other than the buffer from flowing back to the buffer supply source 421 is provided, and further on the downstream side, a three-way electromagnetic valve 423 (V _ba ). The three-way solenoid valve 423 switches communication between the pipe 424 and the pipe 425 and communication between the pipe 415 and the pipe 425. That is, the pipe 415 communicates with the pipe 425 when the three-way solenoid valve 423 is not energized, and the pipe 424 communicates with the pipe 425 when energized. Thereby, the supply of the buffer to the pipe 425 and the supply of air or milli-Q water can be switched.

An insertion agent supply source 431 that stores an insertion agent as one of the chemical solutions is connected to the pipe 434. A check valve 432 is provided downstream 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 downstream is a three-way electromagnetic valve. 433 (V _in ) is provided. The three-way solenoid valve 433 switches communication between the pipe 434 and the pipe 435 and communication between the pipe 425 and the pipe 435. That is, the pipe 425 is communicated with the pipe 435 when the three-way solenoid valve 433 is not energized, and the pipe 434 is communicated with the pipe 435 when energized. Thereby, the supply of the intercalating agent to the pipe 435 and the supply of air, milli-Q water or buffer can be switched.

  As described above, by controlling the two-way solenoid valve 403 and the three-way solenoid valves 413, 423, and 433 in the air and chemical solution supply system, air supplied to the chip cartridge 11 via the pipe 435, milli-Q water, It is possible to switch the supply of chemical solutions such as buffers and intercalating agents, and to control the air supplied and the flow rate of these chemical solutions.

The three-way solenoid valve 433 _communicates with the upstream side of the pipe 435, and the three-way solenoid valve 441 (V _cbin ) _{communicates with the} downstream side thereof. 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 the bypass pipe 446 when not energized and switches the pipe 435 to the pipe 440 when energized. The three-way solenoid valve 445 switches the bypass pipe 446 to the pipe 450 when not energized and switches the pipe 440 to the pipe 450 when energized. With 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.

The piping 440 includes a two-way solenoid valve 442 (V _1in ), a chip cartridge 11, a liquid sensor 443, a two-way solenoid valve 444 (V _1out ), and a three-way solenoid in order from the three-way solenoid valve 441 toward the downstream side. A valve 445 (V _cbout ) is provided. A flow path 114a corresponding to the feeding system of the chip cartridge 11 communicates with the two-way electromagnetic valve 442 side, and a flow path 114b corresponding to the delivery system of the chip cartridge 11 communicates with the two-way electromagnetic valve 444 side. ing. Thereby, the chemical solution, air, and the like are supplied to the delivery system of the chip cartridge 11 via the pipe 440, and the chemical solution, air, and the like can be discharged from the delivery system of the chip cartridge 11. In addition, the flow rates of chemicals and air in the liquid supply and discharge paths can be controlled by the two-way electromagnetic valves 442 and 444. Further, the liquid sensor 443 can monitor the flow rate of the chemical liquid that flows into the chip cartridge 11 or is discharged from the chip cartridge 11.

The piping 450 includes a two-way solenoid valve 451 (V _vin ), a pressure reducing region 452, a two-way solenoid valve 453 (V _out ), a liquid feed pump 454, and three-way in order from the three-way solenoid valve 445 toward the downstream side. A solenoid valve 455 ( _Vww ) is provided. The two-way solenoid valves 451 and 453 prevent the backflow of the chemical solution and air in the path before and after the decompression region 452. Further, the liquid feed pump 454 is a tube pump and is characterized in that it is provided in a discharge system on the delivery side (downstream side) when viewed from the chip cartridge 11. That is, the use of a tube pump is preferable from the viewpoint of preventing contamination because the chemical solution does not contact any mechanism other than the tube wall. Further, by supplying and discharging the chemical liquid and air to and from the chip cartridge 11 by a suction operation, the chemical liquid and the air can be replaced smoothly in the chip cartridge 11, and in the unlikely event, piping is used. No liquid leakage occurs even if the chip cartridge 11 is loosened or the chip cartridge 11 is detached from the pipe 440. Thereby, the safety | security of apparatus installation improves.

  Of course, a pump may be provided in a pipe on the upstream side of the chip cartridge 11, and air or a chemical solution may be pushed out to the chip cartridge 11 by this pump. The pump is not limited to a tube pump, and a syringe pump, a plunger pump, a diaphragm pump, a magnet pump, or the like can also be used.

  The three-way solenoid valve 455 switches so that the pipe 450 communicates with the pipe 461 when not energized and communicates 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, chemical liquids 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 electromagnetic valve 455, and the intercalating agent can be guided to the intercalating agent waste liquid tank 464. This makes it possible to collect and collect the intercalating agent.

  The solenoid valves may be connected by piping such as a Teflon (registered trademark) tube, but in this embodiment, the solenoid valves and flow paths are respectively connected upstream and downstream of the chip cartridge 11. It consists of a manifold structure that is an integral structure. Thereby, since the capacity | capacitance in piping becomes small, a required chemical | medical solution amount can be reduced significantly. Further, since the chemical liquid flow in the pipe is stabilized, the reproducibility and stability of the detection result is improved.

  A liquid feeding process for base sequence detection using the liquid feeding system 13 shown in FIG. 19 will be described with reference to the flowchart of FIG.

  First, a hybridization reaction between the DNA probe immobilized on the electrode 21a and the sample is executed in the cell 115 (s21). In the execution of this hybridization reaction, the temperature control mechanism 14 is controlled so that the bottom surface of the chip cartridge 11, that is, the bottom surface of the printed circuit board 22 is about 45 ° C., for example, and held for 60 minutes.

  In parallel with this hybridization reaction, the chemical solution line is set up (s22). Specifically, the bypass pipe 446 is used by controlling the three-way solenoid valves 441 and 445, and the three-way solenoid valve 433 is energized to supply the insert agent from the insert supply source 431 for about 10 seconds, for example. . The three-way solenoid valve 455 is energized, and the intercalating agent from the pipe 450 is accommodated 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 repeatedly for about 5 seconds, for example. Next, only air is introduced from the pipe 435 into 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 repeatedly for about 5 seconds, for example.

  When the start-up of the chemical solution line is completed and the hybridization reaction is completed, the pipe is cleaned (s23). For example, after the temperature of the printed circuit board 22 is set to about 25 ° C. by the temperature control mechanism 14 and the bypass pipe 446 is purged with milli-Q water, air and milli-Q water are alternately used for about 5 seconds, for example. Introduce repeatedly. Next, chip cartridge cleaning is performed (s24). In the chip cartridge cleaning, 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 alternately for about 5 seconds, for example. 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.

  Next, a pipe buffer purge is performed (s25). In the pipe buffer purge, air is first introduced into the bypass pipe 446 so that the buffer and milli-Q water are not mixed. Next, air and a buffer are alternately introduced into the bypass pipe 446 repeatedly for about 5 seconds, for example. Then, the liquid sensor 447 provided in the bypass pipe 446 confirms that the bypass pipe 446 has been replaced with a buffer.

  Next, buffer injection in the chip cartridge is performed (s26). In the buffer injection in the chip cartridge, first, the bypass pipe 446 is switched to the pipe 440, and air and the buffer are alternately introduced into the chip cartridge 11 alternately for about 5 seconds, for example.

  Next, buffer filling of the chip cartridge 11 is performed (s27). In the buffer filling, the buffer is introduced into the chip cartridge 11 while monitoring the state in the chip cartridge 11 with the liquid sensor 443, and the sample is left to stand at 60 ° C. for 30 minutes, for example, to wash unnecessary samples (s28). After the unnecessary sample cleaning step, the piping 440 is switched to the bypass piping 446, and milli-Q water is introduced to clean the inside of the piping (s29). In this in-pipe cleaning, air and milli-Q water are repeatedly introduced alternately, for example, every 5 seconds.

  Next, the inside of the chip cartridge is cleaned (s30). In the cleaning in the chip cartridge, the bypass pipe 446 is switched to the chip cartridge 11, and air and water are repeatedly introduced alternately for about 5 seconds, for example. Thereafter, the liquid sensor 443 is switched to the bypass pipe 446 after confirming that the chip cartridge 11 is filled with milli-Q water.

  Next, the measurement is started. In the measurement, first, an in-pipe insertion agent purge is performed (s31). In this in-pipe intercalating agent purge, the waste liquid is switched to the intercalating agent waste liquid tank 464 while introducing air into the bypass pipe 446. Next, air and an intercalating agent are alternately and repeatedly supplied to the bypass pipe 446 for about 5 seconds, for example, and then the liquid sensor 447 detects whether the bypass pipe 446 has been replaced with the intercalating agent.

  Next, the insertion agent is injected into the chip cartridge 11 (s32). In this step, first, after switching from the bypass pipe 446 to the chip cartridge 11 side, the air and the intercalating agent are repeatedly introduced alternately for about 5 seconds, for example.

  Next, under the monitoring by the liquid sensor 443, the insertion agent is filled into the chip cartridge 11 (s33). Thereafter, measurement is performed (s34).

  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).

  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 each, 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.

  As described above, according to the process shown in FIG. 20 using the liquid feeding system 13 of FIG. 19, in order to efficiently replace the chemical solution, the inside of the pipe is air and chemical solution / air / chemical solution / air. It is possible to send a liquid by creating a sequence in which chemicals flow alternately. By using such a liquid feeding 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 liquid exchange transition state is reduced, and the reproducibility of the final electrochemical characteristics can be improved. Furthermore, shortening of the liquid feeding time and reduction of the amount of the chemical solution can be realized by improving the efficiency of the chemical solution exchange. In addition, since the chemical solution / air sequence liquid feeding can keep the chemical solution concentration in the reaction cell 115 constant, the in-plane uniformity of the current characteristics is improved, that is, the detection reliability is improved.

  Further, as a method of filling the chemical solution into the cell 115, as a countermeasure when bubbles are mixed, the inside of the pipe 440 on the downstream side of the chip cartridge is closed with the two-way electromagnetic valve 444 as the chip cartridge outlet valve closed. A reduced pressure state (with the pump 454 operating, the two-way solenoid valve 451 is controlled to reduce the pressure reduction region 452 and then the two-way solenoid valve 453 is controlled to change the pressure reduction state of the pressure reduction region 452. Therefore, the chemical solution can be introduced into the chip cartridge reaction cell 115 by opening the two-way electromagnetic valve 444.

  Note that the liquid feeding timing shown in FIG. 20 is merely an example, and various changes can be made according to the purpose, object, and conditions of measurement.

  FIG. 21 is a diagram showing a specific configuration of the measurement system 12. In the measurement system 12 shown in FIG. 21, the voltage of the reference electrode 503 is negatively fed back with respect to the input of the counter electrode 502, so that a desired condition can be obtained in the solution regardless of changes in various conditions such as the electrode in the cell 115 and the solution. This is a three-electrode type potentiostat 12a that applies a voltage of 2.

  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 changes the oxidation current of the intercalating agent. taking measurement.

  The working electrode 501 is an electrode to 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 supplies a current into the cell 115 by applying a predetermined voltage to the working electrode 501. The reference electrode 503 is an electrode that negatively feeds back the 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 by the counter electrode 502 is controlled. Thus, highly accurate oxidation current detection can be performed regardless of various detection conditions in the cell 115.

A voltage pattern generation circuit 510 that generates a voltage pattern for detecting a current flowing between the electrodes is connected to the inverting input terminal of the inverting amplifier 512 (OP _c ) for controlling the reference voltage of the reference electrode 503 via the wiring 512b. Yes.

  The voltage pattern generation circuit 510 is a circuit that generates a voltage pattern by converting a digital signal input from the control mechanism 15 into an analog signal, and includes a DA converter.

A resistor R _s is connected to the wiring 512b. The non-inverting input terminal of the inverting amplifier 512 is grounded, and the wiring 502a is connected to the output terminal. 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 the wiring 512a. The wiring 512a, the protection circuit 500 is provided comprising a feedback resistor _{R ff} and a switch SW _f.

  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, the terminal C is connected in parallel to each. Thereby, a voltage can be simultaneously applied to the plurality of counter electrodes 502 by one voltage pattern.

The wiring 502a, the switch SW ₀ performing on-off control of voltage applied to the terminal C is provided.

  The protection circuit 500 provided in the inverting amplifier 512 is configured such that an excessive voltage is not applied to the counter electrode 502. Therefore, an excessive voltage is applied at the time of measurement, and the solution is electrolyzed, so that stable measurement is possible without affecting the detection of the oxidation current of the desired intercalating agent.

The terminal R is connected to the non-inverting input terminal of the voltage follower amplifier 513 (OP _r ) by the wiring 503a. 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. The wiring 513b is provided with a resistor _{Rf, and} is connected between the resistance of the wiring 512b and the intersection of the wiring 512a and the wiring 512b. Accordingly, a voltage obtained by feeding back the voltage of the reference electrode 503 to the voltage pattern generated by the voltage pattern generation circuit 510 is input to the inverting amplifier 512, and the voltage of the counter electrode 502 is changed based on an output obtained by inverting and amplifying such a voltage. Control.

The terminal W is connected to the inverting input terminal of the trans-impedance amplifier 511 (OP _w ) by the wiring 501a. 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. Resistance _{R w} is provided in the wiring 511a. When the voltage at the terminal O on the output side of the trans-impedance amplifier 511 is V _w and the current is I _w , V _w = I _w · R _w . 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, which will be described later, and are subjected to AD conversion, whereby the electrochemical signals from the working electrodes 501 can be acquired 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 the plurality of working electrodes 501. In this case, a signal switching unit for switching wiring from the plurality of terminals W may be provided in the wiring 501a.

The effect of the measurement system 12 using the potentiostat 12a of FIG. 21 will be described in comparison with the case where a conventional potentiostat is used. A conventional potentiostat is shown in FIG. As shown in FIG. 22, the configuration of the conventional potentiostat 12a ′ is almost the same as that of the potentiostat 12a shown in FIG. The difference is that the inverting amplifier 512 is not provided with the protection circuit 500. Voltage _{V refin} at the output terminal I of the voltage pattern generation circuit 510, the voltage at terminal C _{V c,} a voltage of the terminal R and _{V REFOUT.} The negative feedback of the reference electrode _{503, V refout = R f /} R s · V refin is established.

In this case, an example of the voltage V _refin , the switch switching state of the switches SW ₀ and SW _f , the voltage characteristics of the voltage Vc and the voltage V _refout and the switch switching state is shown for the potentiostat 12a in FIG. 23, the potentiostat 12a. FIG. 24 shows “′”.

23, (a) is a voltage waveform of the voltage V _refin , (b) is a switch switching state of the switch SW ₀ , (c) is a switch switching state of the switch SW _f , (d) is a voltage waveform of the voltage V _c , (E) is a voltage waveform of the voltage _Vrefout .

_{24A is} a voltage waveform of the voltage V _refin , FIG. 24B is a switch switching state of the switch SW ₀ , FIG. _{24C is} a voltage waveform of the voltage V _c , and FIG. _{24D is} a voltage waveform of the voltage V _refout. .

  A measurement method in the conventional potentiostat 12a 'will be described with reference to FIG.

For example, as shown in FIG. 24A, a voltage pattern is generated such that a constant voltage is applied from time t ₁ to time t _{3 and} then the voltage is linearly decreased so as to become voltage 0 at time t _4. Generated by circuit 510. Then, for example, as shown in FIG. 24B, it is assumed that the switch SW ₀ is closed and a voltage is applied to the counter electrode 502 at a time t ₂ when a predetermined time has elapsed from the time t ₁ . In this case, at the start of the measurement, that is until you close the switch SW ₀ is in a state where the switch SW ₀ is opened.

Since the gain of the inverting amplifier 512 is very large, if some voltage is applied to the inverting input terminal of the inverting amplifier 512 before the switch SW ₀ is turned on and the feedback loop is formed, the output of the inverting amplifier 512 is It becomes saturated. On the other hand, even if the voltage V _refin is 0 V, the input offset voltage of the inverting amplifier 512 is saturated. In this case, it saturates to the opposite polarity of the input offset voltage.

As described above, 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, when the switch SW ₀ is closed, an excessive voltage is applied to the counter electrode 502. This excessive 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, it adversely affects the measurement of the intended electrochemical reaction.

In order to eliminate the disadvantages of the conventional potentiostat 12a ', the protection circuit 500 is used in the potentiostat 12a of the present embodiment. For potentiostat 12a of this embodiment, before starting the measurement, i.e. in the initial state before the time _{t a,} the voltage _{V refin} 0V, the switch SW _f closed, and an open state the switch SW ₀ To do. First, the switch SW ₀ in the closed state at time _{t a.} In this state, the switch SW _f is closed and the protection circuit 500 is functioning. Since the inverting amplifier 512 is always used with negative feedback, an excessive voltage is not applied to the counter electrode 502.

At a time t _b after _a predetermined time from the time ta, the switch SW _f is opened and the protection circuit 500 is released. Thereafter, the voltage V _refin generated by the voltage pattern generation circuit 510 from time t ₁ is applied. Since the desired voltage is set to the reference electrode 503 by the voltage V _refin , the response has a first-order lag characteristic, and the counter electrode 502 is not excessively _charged .

  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 a negative characteristic, whereas in the case of the potentiostat 12a of the present embodiment, the voltage becomes negative. However, 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, bubbles are generated in the electrode or the composition of the electrode is changed. On the other hand, by providing the protection circuit 500 as in the example of this embodiment, it is possible to prevent an unintended voltage from being applied to the counter electrode 502. Can be avoided. Accordingly, stable measurement is possible without affecting the detection of the oxidation current of the desired intercalating agent.

  Modification examples 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 type potentiostat is used as the measurement system 12, and FIGS. 28 and 29 show examples in which a four-electrode type potentiostat is used as the measurement system 12.

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 thereof is omitted. The potentiostat 12b is different from the potentiostat 12a in that the protection circuit 500 including the wiring 512a is not provided. Instead of the protection circuit 500, the resistor _{R c} are provided on the wiring 502a. Thus, by connecting the resistor R _c in series with the output of the counter electrode 502 side of the inverting amplifier 512, the voltage applied to the counter electrode by an electric double layer capacitor becomes a first-order lag. Thereby, the influence with respect to the chemical | medical solution in the cell 115 can be decreased.

The potentiostat 12c shown in FIG. 27 is slightly different in configuration from the potentiostats 12a and 12b. In the potentiostat 12c, it provided a current detection resistor R _c to the counter electrode 502 side, and converts the voltage detection current at high input impedance differential amplifier 520. Hereinafter, the configuration will be described in more detail.

As shown in FIG. 27, a voltage pattern generation circuit 510 that generates a voltage pattern for detecting a current flowing between the electrodes is connected to the inverting input terminal of the inverting amplifier 512 (OP _c ) via a wiring 512b. Resistor _{R s} is connected to the wiring 512b. The non-inverting input terminal of the inverting amplifier 512 is grounded, and the wiring 512f is connected to the output terminal. The output terminal and the inverting input terminal of the inverting amplifier 512 are connected by the protection circuit 500.

Wiring 512f switch SW ₀ is provided to perform on-off control of voltage applied to the terminal C. Further, 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.

The wiring 521b is a current detection resistor _{R c} are provided. Further, the wiring 521b branches to a wiring 502a and a wiring 521e at an intersection 521d. The wiring 502 a is connected to the terminal C, and the wiring 521 e is connected to the non-inverting input terminal of the amplifier 523 in the high input impedance differential amplifier 520.

The configuration of the voltage follower amplifier 513, the wirings 513a and 513b, and the resistor _Rf for feeding back the voltage from the terminal R on the reference electrode 503 side to the inverting input terminal of the inverting amplifier 512 is the same as that in FIG.

  The terminal W on the working electrode 501 side is grounded by the wiring 501a.

High input impedance differential amplifier 520 outputs an output from the wiring 521a not through the current detection resistor R _c, amplifies the differential voltage of the output wiring 521e passing through the current detection resistor R _c to the terminal O. Each of the inverting input terminal of the amplifier 522 and 523 are connected by wire 522a having a resistance _{R 1.} Inverting input terminal and the output terminal of the amplifier 522 is connected by a wiring 522b having a resistance _{R 2.} Inverting input terminal and the output terminal of the amplifier 523 is connected by a wire 523a having a resistance _{R 3.} The output of amplifier 522 is connected to the inverting input terminal of the amplifier 524 via the resistor R _4. The output of amplifier 523 is connected to the non-inverting input terminal of the amplifier 524 through the resistor R _5. The non-inverting input terminal of the amplifier 524 is grounded via a resistor R _6. Inverting input terminal and the output terminal of the amplifier 524 is connected by a wiring 522d having a resistance _{R 7.} The output terminal of the amplifier 524 is connected to the terminal O by a wiring 524b.

  In the case of this potentiostat 12c, the oxidation current is detected not from the working electrode 501 but from the counter electrode 502 side.

  Thus, even when the potentiostat 12c as shown in FIG. 27 is used, the same effect as the potentiostat 12a can be obtained.

  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 the same as the configuration of the potentiostat 12a of FIG. In the case of the potentiostat 12d, the voltages from the two reference electrodes 5031 and 5032 are 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. In this way, 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.

As shown in FIG. 28, the terminal R ₁ on the reference electrode 5031 side is connected to the non-inverting input terminal of the amplifier 523. A terminal R ₂ on the reference electrode 5032 side is connected to a non-inverting input terminal of the amplifier 522. The high input impedance differential amplifier 520 differentially amplifies and outputs two voltages at the non-inverting input terminals of each of the amplifiers 522 and 523. The output side is connected to the wiring 512b via a resistor _Rf .

  Thus, even if the potentiostat 12d shown in FIG. 28 is used, the same effect as the potentiostat 12a can be obtained.

  The four-electrode potentiostat 12e shown in FIG. 29 has the same basic configuration as the potentiostat 12c shown in FIG. What is different from the potentiostat 12c is that the potentiostat 12e has two reference electrode extraction voltages, and the two voltages are differentially amplified and fed back to the counter electrode 502 side. Since the configuration of the counter electrode 502 side and the working electrode 501 side is the same as that of the potentiostat 12c, detailed description thereof is omitted. Reference numeral 520 'denotes a high input impedance differential amplifier having the same configuration as that of the high input impedance differential amplifier 520 described above.

As shown in FIG. 29, potentiostat 12e includes two reference electrode 5031 side terminal _{R 1} of the non-inverting non-inverting input terminal and the amplifier 522 of the output each reference electrode 5032 side terminal _{R 2} amplifier 523 Connect to the input terminal. As described above, the high input impedance differential amplifier 520 differentially amplifies these two inputs and outputs them. A resistor _Rf is connected to the output side, and the resistor _Rf is connected to the wiring 512b via the resistor _Rf . As a result, the output of the high input impedance differential amplifier 520 is fed back to the inverting input side of the inverting amplifier 512.

  FIG. 30 is a conceptual diagram showing the relevance of 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 / received to / from the plurality of control mechanisms 15 through 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 measurement unit 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.

  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 15 a includes 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.

  The initial value register 151, the step value register 152, the end value register 153, the interval register 154, and the operation setting register 155 store the initial value, step value, end value, measurement time interval, and operation mode that can be set by the main processor 16a, respectively. To do. When these initial value, step value, end value, measurement time interval, and operation mode are set, the data measurement operation is started.

The initial value, the step value, and the end value indicate values corresponding to the voltage value 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. The For example, when a voltage pattern having a predetermined waveform is generated from time t ₁ to time t _5, the voltage value at time t ₁ corresponds to the initial value, and the voltage value is incremented from the time t ₁ at every measurement time interval Δt. Fluctuates, and such a voltage value continues to be ticked to the end value.

  The selector 158 selects and outputs the initial value only at the start of measurement out of 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. The voltage pattern generation circuit 510 generates a voltage having a voltage value corresponding to the output value from the selector 158. Thereby, the voltage pattern of the voltage waveform shown in FIG. 23A can be generated.

  The addition register 157 temporarily stores the output value of the selector 158 in synchronization with the output signal of the timing generator 161.

  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 outputs a value corresponding to the voltage value obtained by adding the step value to the output voltage value to the measurement system 12. Output. The comparator 159 compares the addition result of the adder 156 with the end value from the end value register 153, and outputs a signal indicating the end of counting to the counter 160 when the addition result exceeds the end value.

  The counter 160 continues to count the clock until the end of the count is input from the comparator 159 based on the operation setting mode from the operation setting register 155 for the 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 4-pole setting mode, an 8-pole setting mode, etc. can be set according to the number of working electrodes simultaneously measured. 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, the measurement time interval is counted every four time periods and the count value is output to the timing generator 161. Thus, when the multi-pole setting mode is set, the measurement time interval is counted for each time period divided by the number of poles.

  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 the clock. 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.

  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 from the intercalating agent can be detected simultaneously 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.

  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 AD signal to the data memory 15 b via the data bus 164. As a result, every time a write signal from the timing generator 161 is input, data from the data bus 164 can be sequentially written into the data memory 15b at an address position given for each write signal.

  For example, in the unipolar 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 15b once every 10 msec, and the signal detector 162 outputs a data bus. One digital conversion value of the electrochemical signal is output to the data memory 15b via 15b.

  In the case of the 4-pole setting mode, if the measurement time interval is 10 msec, the write signal and four addresses are output from the timing generator 161 to the data memory 15b four times at 10 msec, and the signal detector 162 outputs the data bus 15b. Then, four digital conversion values of the electrochemical signal are sequentially output to the data memory 15b. Thereby, the electrochemical signal detected almost simultaneously at every measurement time interval can be stored as data.

  In order to improve the measurement accuracy, in 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 in a short time within the measurement time interval, it is possible to maintain measurement accuracy that is not affected by the measurement time interval. For example, if the measurement time interval is 10 msec, the timing generator 161 does not generate a switching signal until the first 9 msec, but generates four switching signals in 1 msec from 9 msec to 10 msec and outputs them to the signal switching unit 163. Program. Thereby, 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, and high accuracy can be maintained.

  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.

  In this way, the signals of the working electrode 501 can be measured almost simultaneously by selectively detecting the plurality of measured electrochemical signals by switching the timing generator 161 in a shorter time than the measurement time interval. it can.

  Next, an example of a measurement data analysis method for performing signal analysis by the computer 16 based on the measurement data will be described. Here, a type determination analysis method 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 shown in the flowchart of FIG. I will explain. Although not particularly shown in FIGS. 1 and 30, the main processor 16a of the computer 16 executes an analysis program including a plurality of commands for performing type determination filtering, type determination processing, determination result output, and the like. Thus, type determination filtering, type determination processing, and determination result output are executed. A control program is separately provided for the control of the control mechanism 15 described above. These analysis programs and control programs 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. May be read and executed.

  As a premise for performing the measurement data analysis, first, four types of target base sequences to be detected are prepared as bases at SNP positions as A, G, C, and T, and base sequences complementary to the target base sequences are prepared. A plurality of target complementary DNA probes are immobilized on each working electrode 501 for each type. In addition, a plurality of DNA probes having base sequences different from those of these four types of target complementary DNA probes (hereinafter referred to as negative controls) are immobilized on another working electrode 501 (s61). In principle, the number of DNA probes immobilized on the working electrode 501 is one.

  Next, a sample containing the sample DNA probe is injected into the base sequence detection chip on which the target complementary DNA probe is immobilized to cause an electrochemical reaction such as a hybridization reaction (s62). After that, the representative current value is calculated using the measurement system 12 (s63).

  The representative current value is a numerical value effective for quantitatively grasping the occurrence of the hybridization reaction of each DNA probe. As an example, the maximum value (peak current value) of the detected signal current value, etc. 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 the background current other than the oxidation current signal from the intercalating agent.

  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 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 or voltage, and the like can be determined as the representative value.

Measurement data relating to the target DNA in which the base at the SNP position is A, G, C, T type, that is, representative current values are defined as X _a , X _g , X _c , and X _t , respectively. Define the value as _Xn . Since a plurality of representative current values are obtained according to various types, the first X _a is defined as X _a1 , the second X _a is defined as X _a2 ,...

Moreover, the base of SNP positions A, G, C, the number of _n a representative current value obtained of the target DNA was a _{T-type, n g, n c, n} t pieces, the representative current value obtained for the negative control the number is defined as _{n n} number.

Next, a type determination filtering process is executed to remove apparently abnormal data from the obtained representative current values X _a , X _g , X _c , X _t , and X _n (s64).

FIG. 33 shows a flowchart of this type determination filtering process. The type determination filtering process of FIG. 33 is performed separately for X _a , X _g , X _c , X _t , and X _n . For example, taking X _a as an example, among the n _a number of representative current value obtained for X _a, eliminates the representative current value appears clearly abnormal data in this type determination filtering. _{X g, X c, X t} , is similarly performed for _{X n.}

In the description of FIG. 33, since the same process in accordance with the data type is performed, illustrating the filtering of X _a as an example.

Specifically, as shown in FIG. 33, first, all measurement data of the measurement group is set, that is, the data set is set (s81). For example, in the case of _{X a, X a1, X a2} , ..., to set the _{X ana} as a data set.

Next, a CV value (hereinafter, CV ₀ ) is calculated for these measurement data X _a1 , X _a2 ,..., X _ana (s82). This CV ₀ is obtained by dividing the standard deviation of the measurement data X _a1 , X _a2 ,..., X _ana by the average value. Then, it is determined whether or not the obtained value CV ₀ is 10%, that is, 0.1 or more (s83).

If it is 10% or more, the CV value (hereinafter referred to as CV ₁ ) of na-1 data set excluding the minimum value of the measurement data is calculated (s84). If it is less than 10%, it is determined that there is clearly no abnormal data, and the process proceeds to type determination described later.

After calculating CV ₁ , it is determined whether CV ₀ ≧ 2 × CV ₁ (s85). If this inequality holds, the process proceeds to (s86), and na-2 data sets excluding the minimum value of the measurement data are newly defined as data sets, and the process returns to (s82) to filter abnormal data. Repeat.

If the inequality does not hold, it is determined that there is abnormal data on the maximum value side instead of the minimum value side, and the CV values (hereinafter referred to as CV ₂ ) of na-2 data sets excluding the maximum value among the measurement data. Is calculated (s87). Then, it is determined whether CV ₀ ≧ 2 × CV ₂ is satisfied (s88). If it is established, na-3 data sets excluding the maximum value from the measurement data are newly defined as data sets, and the process returns to (s82), and abnormal data filtering is repeated. If not established, it is determined that there is no apparently abnormal data, and the process proceeds to type determination described later.

The type determination filtering described above is also performed for X _g , X _c , X _t , and X _n .

  Next, a type determination process is executed using the obtained type determination filtering result (s65). An example of this type determination process will be described with reference to the flowchart of 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 process is roughly divided into a maximum group determination algorithm and a two-sample t-test algorithm.

As shown in FIG. 34, first, an average value of representative current values for each group is extracted (s91). The groups are different groups such as X _a , X _g , X _c , X _t , X _n, etc. with different target base sequences, and those with the same target base sequence are the same group. In (s64), measurement data from which abnormal data is clearly excluded by type determination filtering is extracted. Of course, measurement data from which data has been excluded by filtering other than the type determination filtering in (s64) may be extracted, or measurement data that is not filtered at all may be extracted. Instead of the average value of the representative current values, another statistical processing value obtained by statistical processing from these statistical values may be obtained.

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 average value obtained _{X a, X g, X c} , X t, the _{X n} in each _group, and _{M a, M g, M c} , M t, M n.

Next, regarding the obtained average values M _a , M _g , M _c , M _t , and M _n , it is determined whether or not the maximum is the average value M _g of the group G (s92). If it is the maximum, the process proceeds to (s93), and if not, the process proceeds to (s97).

In (s97), for the average values M _a , M _g , M _c , M _t , and M _n , it is determined whether or not the maximum is the average value M _t of the group T. If it is the maximum, the process proceeds to (s98), and if it is not the maximum, neither the group G nor T is the maximum, and the reexamination is performed because the determination is impossible.

In (s93), it is determined whether or not there is a difference between the measurement data X _g1 , X _g2 ,... Of the group G and the measurement data X _n1 , X _n2,. For example, a two-sample t-test is used to determine whether there is a difference. Specifically, the representative relationship between the probability P obtained by the two-sample T-test and the significance level α is compared,
H0: No significant difference if P ≧ α (null hypothesis)
If H1: P <α, there is a significant difference (alternative hypothesis)
Is determined. The user can set the significance level α using the computer 16. In the example of (s93), the question H1 is asked whether there is a difference between the measurement data of the group G and the measurement data of the group N. If there is no difference between these two groups, The hypothesis of H0 to be assumed is set. Then, the probability is obtained assuming that the difference between the two groups is summarized in the average value M _g of the measurement data of group G and the average value M _n of the measurement data of group N. The probability is calculated by calculating the statistical constant t and the degree of freedom φ based on the statistical values X _g1 , X _g2 ,... Of the group G and the statistical values X _n1 , X _n2,. The probability P is obtained from the integral value.

  For 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.

  When it is determined that the determination result is “difference” in this way, the process proceeds to (s94), and when it is determined that there is no difference, re-examination is performed as determination is impossible.

  In (s94), it is determined whether there is a difference between the two groups using the two-sample t-test similar to (s93) for group G and group A. If there is a difference, the process proceeds to (s95).

  In (s95), it is determined whether there is a difference between the two groups using the two-sample t-test similar to (s93) for group G and group C. If there is a difference, the process proceeds to (s96).

  In (s96), it is determined whether or not there is a difference between the two groups using the two-sample t-test similar to (s93) for group G and group T. If there is a difference, the group G type is determined. This is because the group G type has a maximum average value and is different from other measurement groups. If there is no difference, the group GT type is determined. This is because the group G type has the maximum average value, but there is no difference in measurement results between the group G type and the group T type.

  In (s98), it is determined whether there is a difference between the two groups using the two-sample t-test similar to (s93) for group T and group N. If there is a difference, the process proceeds to (s99).

  In (s99), it is determined whether there is a difference between the two groups using the two-sample t-test similar to (s93) for group T and group A. If there is a difference, the process proceeds to (s100).

  In (s100), it is determined whether there is a difference between the two groups using the two-sample t-test similar to (s93) for group T and group C. If there is a difference, the process proceeds to (s101), and if there is no difference, the determination is impossible and the inspection is performed again.

  In (s101), it is determined whether there is a difference between the two groups using the two-sample t-test similar to (s93) for group T and group G. If there is a difference, the group T type is determined. This is because the group T type has a maximum average value and is different from other measurement groups. If there is no difference, the group GT type is determined. This is because the group T type has the maximum average value, but there is no difference in measurement results between the group T type and the group G type.

  The above determination results are 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.

  In FIGS. 32 to 34, a method of determining which of G type, T type, and GT type is shown, but any two of A type, G type, C type, and T type are shown. Of course, it can be applied to the determination of types or their heterogeneity. In addition, it is not always necessary to acquire measurement data for four types of groups of A type, G type, C type, and T type, and it may only be acquired for two groups related to two possible bases of the SNP. One group of negative controls may be added to two groups.

  An automatic base sequence analysis method using the base sequence detection apparatus described above will be described with reference to the sequence diagram of FIG.

  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 parameter is a control parameter for controlling the control mechanism 15. The control parameters used in the control mechanism 15 are a measurement system control parameter for controlling the measurement system 12, a liquid feed system control parameter for controlling the liquid feed system 13, and a temperature control mechanism for controlling the temperature control mechanism 14. Consists of control parameters.

  The measurement system control parameters are input setting parameters stored in the initial value register 151, the step value register 152, the end value register 153, the interval register 154, and the operation setting register 155 shown in FIG. , End value, measurement time interval, and operation mode.

  The liquid feed system control parameter controls the electromagnetic valves 403, 413, 423, 433, 441, 442, 444, 445, 451, 453, and 463 shown in FIG. It has a sensor control parameter and a pump control parameter for controlling the pump 454. These solenoid valve control parameter, sensor control parameter, and pump control parameter are the control amount and control target of the control target as conditions for sequentially executing a series of steps as shown in (s22) to (s36) of FIG. The control timing, control conditions for controlling the control target, and the like are included as parameter details.

  In principle, the temperature control parameter is given accompanying the liquid feeding system control parameter. That is, by setting the liquid supply system control parameter, the temperature control parameter is set corresponding to the operation of the liquid supply system 13. Thereby, the temperature control of the temperature control mechanism 14 interlock | cooperated with the liquid feeding system 13 is attained.

  By executing the automatic analysis, the automatic analysis condition parameter is 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 delivery system 13 based on the liquid delivery system control parameter, and controls the temperature based on the temperature control mechanism control parameter. The control mechanism 14 is controlled. 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 the 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, but a typical example will be described with reference to FIG.

  In addition to this automatic analysis, the user prepares the chip cartridge 11. First, a printed circuit board 22 sealed with a base sequence detection chip 21 on which a desired DNA probe is fixed to a working electrode 501 is fixed to a support 111 of a chip cartridge 11 by a substrate fixing screw 25, and then transferred to the chip cartridge 11. Is attached (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. (S402). A sample is injected from the sample injection port 119 into the chip cartridge 11 (s403). The hybridization reaction (s21) is started by attaching the chip cartridge 11 to the apparatus main body and performing a start operation. Note that the volume of the sample to be injected is preferably slightly larger than the volume of the cell 115. Thereby, the inside of the cell 115 can be completely filled with the sample without air remaining.

  The control mechanism 15 starts controlling the timing of the measurement system based on the measurement system control parameter received from the computer 16 (s303).

  Further, the control mechanism 15 sequentially controls each component of the liquid feeding system 13 based on the liquid feeding system control parameter received from the computer 16 (s304). Although not particularly shown in FIG. 35, temperature control of the temperature control mechanism 14 is performed based on the temperature control mechanism control parameter in conjunction with the control of the liquid feeding system 13. By this control, the liquid feeding system 13 automatically executes the liquid feeding process including the hybridization reaction shown in (s21) to (s36) (excluding s34) in FIG. 20 (s305) and is designated in the liquid feeding process. The temperature control mechanism 14 is automatically controlled so that the base sequence detection chip 21 is set to the set temperature.

  The control mechanism 15 issues a measurement command to the measurement system 12 in synchronization with the timing of the measurement process (s34) in the middle of the liquid feeding process (s305). That is, at the timing of the measurement step (s34) of the liquid feeding step, the initial value, the step value, and the initial value register 151, the step value register 152, the end value register 153, the interval register 154, and the operation setting register 155 of the control mechanism 15 Stores the end value, measurement time interval, and operation setting mode. Note that the above-described measurement system timing control in (s303) may be performed simultaneously with this (s305).

  Based on the measurement command, the measurement system 12 generates a voltage pattern, for example, to perform measurement (s306), and the obtained measurement signal is output from the terminal O to the control mechanism 15 (s307). The control mechanism 15 processes the received measurement signal and stores it as measurement data in the data memory 15b (s308). The measurement data is output to the computer 16 via the local bus 17 (s309). The computer 16 receives this measurement data (s310).

  When necessary measurement data is obtained in this way, the computer 16 executes type determination filtering of (s64) shown in FIG. 33 based on the measurement data. When the type determination filtering ends, the type determination process shown in FIG. 34 is executed based on the filtered data (s65). Finally, the obtained determination processing result is displayed on a display device provided in the computer 16 (s66).

  Thus, according to this embodiment, since the shape of the cell 115 is optimized, the in-plane uniformity within the cell 115 can be improved.

  In addition, after injecting the sample DNA solution into the chip cartridge 11, from hybridization, washing of non-specifically adsorbed DNA with a buffer solution, injection of an intercalating agent, electrochemical measurement, storage of measurement data, target base sequence based on the measurement data Up to this determination can be performed automatically. Thereby, the reproducibility and detection accuracy of the detection signal can be improved, and the time until the result can be shortened.

  The present invention is not limited to the above embodiment.

  Although the probe immobilized on the working electrode 501 is a DNA probe, it may be a probe made of a nucleic acid other than DNA, or may be a probe having a predetermined base sequence other than a nucleic acid.

  The sharing of processing between the computer 16 and the control mechanism 15 is not limited to that described above. For example, if the measurement system 12, the liquid supply system 13, and the temperature control mechanism 14 have a processor that interprets commands 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.

  If the measurement system 12, the liquid supply system 13, and the temperature control mechanism 14 have a processor that manages the timing, the management of the measurement system 12, the liquid supply system 13, and the temperature control mechanism 14 is managed by that processor. Each process is executed based on the timing to perform. 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, the timing need not be managed.

  Further, the computer 16 may perform timing control of the measurement system 12, the liquid supply system 13, the temperature control mechanism 14, and the control mechanism 15.

  Moreover, although the sample injection port 119 showed the example connected with the sending port 116b, you may make it connect with the sending port 116a. Moreover, although the case where the number of the input ports 116a and the number of the output ports 116b is one each is shown, it is not limited to this. For example, the number of ports may be increased to any number, for example, one input port 116a is provided at the center of the cell 115, and one output port 116b is provided at a plurality of radial positions on the periphery of the cell 115. Furthermore, although the example in which the sending port 116a and the sending port 116b are extended perpendicularly | vertically with respect to the cell bottom face was shown, it is not limited to this, It is the structure extended in parallel with respect to the cell bottom face. Also good. Moreover, although the electrodes 21a and 21b and the bonding pad 221 on the base sequence detection chip 21 are shown as a laminated structure of Ti or Au, electrodes or pads using other materials may be used. 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.

  The liquid feeding system 13 is not limited to that shown in FIG. For example, a more complicated reaction in the cell 115 can be executed by adding a supply system that supplies chemicals and gases other than air, milli-Q water, buffer, and intercalating agent according to the type of reaction. Moreover, you may perform control of the chemical | medical solution of each piping, the supply path | route of air, and supply amount other than a solenoid valve. The operation of the liquid delivery system 13 shown in FIG. 20 is only an example, and various changes can be made according to the purpose of the reaction.

  Moreover, although the case where this base sequence automatic analyzer 1 is used for a type | mold determination was shown in FIGS. 32-34, it is only an example and may be used for the other analysis objective. Further, the automation method shown in FIG. 35 is only an example, and the automation sequence can be changed by changing the configuration of the chip cartridge 11, the measurement system 12, the liquid feeding system 13, the temperature control mechanism, and the control mechanism 15. Is done.

  Although the case where all of the working electrode 501, the counter electrode 502, and the reference electrode 503 are arranged on the base sequence detection chip 21 is shown, the present invention is not limited to this. For example, only the working electrode 501 may be disposed 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. Either the counter electrode 502 or the reference electrode 503 may be detected by the base sequence. You may arrange | position on the chip | tip 21. FIG. As described above, the working electrode 501, the counter electrode 502, and the reference electrode 503 may be three-dimensionally arranged on different planes.

  Further, the relationship between the base sequence detection chip 21 and the chip cartridge upper lid 112 side may be used upside down.

  An example in which SNPs detection is performed using the base sequence detection device of the above-described embodiment will be described below. Here, it is applied when determining whether the SNPs base sequence of the MxA-88 position gene is G / G type, T / T type, or G / T heterozygous.

  A DNA probe having a sequence complementary to the MxA gene is immobilized in advance on the working electrode 501 of the base sequence detection chip. 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 (referred to as negative control) are immobilized on different 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 the working electrode 501 made of Au. In this way, the printed circuit board 22 on which the prepared base sequence detection chip 21 is sealed is mounted on the chip cartridge 11.

  Next, a target DNA whose base at the SNP position is G-type is dissolved in 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 116 a is formed at a position in contact with the sealing material 24, the sample solution can be completely filled in the cell 115 without leaving bubbles in the cell 115.

  Next, the chip cartridge 11 is mounted on the apparatus main body (measurement system 12, liquid feeding system 13, temperature control mechanism 14), and the apparatus 16 is started by the computer 16, whereby all subsequent processing is automatically performed. Is called.

  Here, the contents of the automatic processing will be described. First, a reaction (hybridization) is performed at 45 ° C. for 15 minutes. Thereafter, the 0.2xSSC-1 mmol / L EDTA solution is fed into the cell 115 by controlling the electromagnetic valve and pump of the liquid feeding system 13. Then, the DNA adsorbed nonspecifically to the electrodes 211 and 212 having different sequences on the base sequence detection chip 21 is washed by holding the solution in the cell 115 for 30 minutes at 55 ° C. Next, 10 μmol / L Hoechst 33258 solution is fed into the cell 115. Then, the oxidation current from Hoechst 33258 in each working electrode 501 is measured by the measurement system 12 in a state where the cell 115 is filled.

  Subsequently, the computer 16 extracts a region corresponding to the Hoechst oxidation current from the current / voltage characteristic curve by an analysis program, and derives the peak current value for each electrode (working electrode 501). Furthermore, statistical processing such as type determination filtering is performed according to the algorithm of the analysis program to perform type determination of the target DNA. The obtained determination result is displayed on the display of the computer 16. As a result, the signal intensity from the electrode corresponding to the probe 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.

  The uniformity of the current value for the same type of electrode in the surface of the base sequence detection chip 21 was within 3% in terms of the CV value. As a result, the reliability of SNP detection is improved as compared with the conventional method.

The conceptual diagram which shows the whole structure of the base sequence detection apparatus which concerns on one Embodiment of this invention. The figure which shows the detail of a structure of the chip cartridge which concerns on the same embodiment The figure which shows the support body and chip cartridge upper cover before fixing with the upper cover fixing screw which concerns on the same embodiment. The figure which shows the detailed structure of the printed circuit board which mounted the base sequence detection chip concerning the embodiment. The figure which shows the chemical | medical solution supply system which leads to the cell which concerns on the same embodiment, and a cell. The figure which shows the modification of the cell which concerns on the same embodiment. The figure which shows the more detailed structure of the positional relationship of each component vicinity of the cell which concerns on the same embodiment. The figure which shows the modification of each component of the cell vicinity which concerns on the same embodiment. Sectional drawing of the modification of the shape of the cell which concerns on the embodiment. Sectional drawing of the modification of the shape of the cell which concerns on the embodiment. Sectional drawing of the modification of the shape of the cell which concerns on the embodiment. Sectional drawing of the modification of the positional relationship of the cell which concerns on the embodiment, and a liquid feeding port and a liquid discharge port. Sectional drawing of the modification of the positional relationship of the cell which concerns on the embodiment, and a liquid feeding port and a liquid discharge port. Sectional drawing of the modification of the positional relationship of the cell which concerns on the embodiment, and a liquid feeding port and a liquid discharge port. Sectional drawing of the modification of the positional relationship of the cell which concerns on the embodiment, and a liquid feeding port and a liquid discharge port. Sectional drawing of the modification of the positional relationship of the cell which concerns on the embodiment, and a liquid feeding port and a liquid discharge port. Process sectional drawing of the manufacturing method of the base sequence detection chip and printed circuit board concerning the embodiment. The top view of the base sequence detection chip concerning the embodiment. The figure which shows an example of the specific structure of the liquid feeding system which concerns on the embodiment. The figure which shows the flowchart of the liquid feeding process for the base sequence detection using the liquid feeding system which concerns on the same embodiment. The figure which shows the specific structure of the measurement system which concerns on the same embodiment. The figure which shows the structure of the conventional potentiostat. The figure which shows the voltage characteristic which concerns on the same embodiment. The figure which shows the voltage characteristic of the conventional potentiostat. The figure which shows the current / voltage characteristic curve applied to the counter electrode in the potentiostat which concerns on the same embodiment, and the conventional potentiostat. The figure which shows the modification of the potentiostat which concerns on the same embodiment. The figure which shows the modification of the potentiostat which concerns on the same embodiment. The figure which shows the modification of the potentiostat which concerns on the same embodiment. The figure which shows the modification of the potentiostat which concerns on the same embodiment. The conceptual diagram which shows the relationship with the control mechanism which concerns on the embodiment, and the other component of a computer. The figure which shows an example of the detailed structure of the control mechanism which concerns on the same embodiment. The figure which shows an example of the measurement data analysis method which concerns on the same embodiment. The figure which shows the flowchart of the type determination filtering process which concerns on the same embodiment. The figure which shows an example of the type | mold determination process which concerns on the same embodiment. The sequence diagram of the base sequence automatic analysis method using the base sequence detection apparatus which concerns on the same embodiment. The figure which shows an example of the optimal cell shape which concerns on the same embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Base sequence detection apparatus, 10 ... Measurement unit, 11 ... Chip cartridge, 12 ... Measurement system, 12a, 12b, 12c, 12d, 12e ... Potentio stat, 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 ... electric connector, 23 ... sealed Stop resin, 24, 24a ... Sealing material, 25 ... Substrate fixing screw, 110 ... Chip cartridge body, 111 ... Support, 112 ... Chip cartridge upper cover, 113a, 113b ... Interface section, 114a, 114b ... Flow path, 115 ... Cell 115a, 115b ... cell hole, 115c, 115e, 115f, 115g, 115h ... straight line, 115 ... Counterbored hole, 116a ... Discharge port, 116b ... Liquid feed port, 117 ... Upper lid fixing screw, 117a ... Screw hole, 119 ... Sample inlet, 120 ... Lid, 121 ... Sealing material, 500 ... Protection circuit, 501 ... Action Pole, 502 ... Counter electrode, 503 ... Reference electrode, 510 ... Voltage pattern generation circuit

Claims (6)

Analysis of the base sequence of a sample by applying a voltage to the counter electrode when a base sequence detector having a working electrode, a counter electrode, and a reference electrode to which a probe having a predetermined base sequence is fixed is mounted. A base sequence automatic analyzer for performing measurement for processing,
A voltage pattern generation circuit for generating a voltage pattern to be applied to the counter electrode;
A feedback circuit that feeds back the voltage of the reference electrode to the counter electrode;
An operational 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;
A resistor provided between an input terminal and an output terminal of the operational amplifier ;
A base sequence automatic analyzer comprising: a first switch that performs on / off control of a current flowing through the resistor. Analysis of the base sequence of a sample by applying a voltage to the counter electrode when a base sequence detector having a working electrode, a counter electrode, and a reference electrode to which a probe having a predetermined base sequence is fixed is mounted. A base sequence automatic analyzer for performing measurement for processing,
A voltage pattern generation circuit for generating a voltage pattern to be applied to the counter electrode;
A feedback circuit that feeds back the voltage of the reference electrode to the counter electrode;
An operational 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;
A resistor provided between the operational amplifier and the counter electrode;
A base sequence automatic analysis device comprising: a switch for performing on / off control of a current flowing through the resistor. Analysis of the base sequence of a sample by applying a voltage to the counter electrode when a base sequence detector having a working electrode, a counter electrode, and a reference electrode to which a probe having a predetermined base sequence is fixed is mounted. A base sequence automatic analyzer for performing measurement for processing,
A voltage pattern generation circuit for generating a voltage pattern to be applied to the counter electrode;
A feedback circuit that feeds back the voltage of the reference electrode to the counter electrode;
An operational 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;
A resistor provided between the operational amplifier and the counter electrode;
A switch for performing on / off control of a current flowing through the resistor;
A high output impedance differential amplifier that amplifies a differential voltage between an output from a wiring connected between the operational amplifier and the resistor and an output from a wiring connected between the resistor and the counter electrode; A base sequence automatic analyzer. A second switch provided between the operational amplifier and the counter electrode;
In the initial state, the input voltage from the voltage pattern generation circuit is set to zero, the first switch is closed, and the second switch is opened, and at the next moment following the initial state, The second switch is switched to the closed state, the first switch is switched to the open state after a predetermined time from the switching of the second switch to the closed state, and the predetermined time from the switching of the first switch to the open state And a control means for controlling the voltage pattern generation circuit, the first switch, and the second switch so that the voltage generated by the voltage pattern circuit is applied to the counter electrode later. The base sequence automatic analyzer according to claim 1. When a base sequence detection device having a working electrode to which a probe having a predetermined base sequence is fixed, a counter electrode, and a plurality of reference electrodes is mounted, the base sequence of the sample is applied by applying a voltage to the counter electrode A base sequence automatic analyzer that performs measurement for the analysis processing of
A voltage pattern generation circuit for generating a voltage pattern to be applied to the counter electrode;
A feedback circuit that differentially amplifies the voltages of the plurality of reference electrodes using a high input impedance differential amplifier and feeds back the output voltage to the counter electrode;
An operational 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;
A resistor provided between an input terminal and an output terminal of the operational amplifier ;
A base sequence automatic analysis device comprising: a switch for performing on / off control of a current flowing through the resistor. When a base sequence detection device having a working electrode to which a probe having a predetermined base sequence is fixed, a counter electrode, and a plurality of reference electrodes is mounted, the base sequence of the sample is applied by applying a voltage to the counter electrode A base sequence automatic analyzer that performs measurement for the analysis processing of
A voltage pattern generation circuit for generating a voltage pattern to be applied to the counter electrode;
A feedback circuit that differentially amplifies the voltages of the plurality of reference electrodes using a high input impedance differential amplifier and feeds back the output voltage to the counter electrode;
An operational 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;
A resistor provided between the operational amplifier and the counter electrode;
A switch for performing on / off control of a current flowing through the resistor;
A high output impedance differential amplifier that amplifies a differential voltage between an output from a wiring connected between the operational amplifier and the resistor and an output from a wiring connected between the resistor and the counter electrode; A base sequence automatic analyzer.

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