JP2016059370A - Base sequence detection chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a base sequence detection chip in which sending of air bubbles to a flow channel on an electrode can be suppressed.SOLUTION: A base sequence detection chip of this embodiment comprises: a plurality of electrode groups separately arrayed on a substrate from each other, in which each electrode group has a plurality of separately arranged first electrodes; probes which are provided corresponding to each first electrode, fixed to a corresponding first electrode, and have a base sequence; and a cover member which is provided on the substrate so as to cover the plurality of electrode groups along an array direction of the plurality of electrode groups, forms a flow channel together with the substrate, and has an inlet into which reagent flows and an outlet from which the reagent having passed the flow channel flows out, in which the first electrode closest to the inlet is provided at a position remote from the inlet by 2.0 times or more the width of the flow channel.SELECTED DRAWING: Figure 9

Description

  Embodiments described herein relate generally to a base sequence detection chip.

  A current detection type DNA sequence detection chip is known. This current detection type DNA sequence detection chip has a configuration in which at least one DNA probe having a known base sequence is arranged on a plurality of electrodes provided on a substrate. DNA probes having different base sequences are arranged on different electrodes.

  Detection of a DNA sequence is performed by flowing a reagent containing an insertion agent through a detection channel formed on an electrode of a DNA sequence detection chip. If bubbles are present on the electrode when this reagent is flowed, accurate current detection cannot be performed, and it is difficult to accurately detect the DNA sequence.

Japanese Patent No. 4729030

  The present embodiment provides a base sequence detection chip capable of suppressing air bubbles from being sent to the channel on the electrode.

  The base sequence detection chip according to the present embodiment includes a plurality of electrode groups arranged on a substrate so as to be spaced apart from each other, and each electrode group includes a plurality of first electrodes arranged apart from each other. And a probe provided corresponding to each first electrode, fixed to the corresponding first electrode and having a base sequence, and the plurality of electrode groups so as to cover the plurality of electrode groups along the arrangement direction of the plurality of electrode groups A cover member provided on a substrate and forming a flow path with the substrate, the cover member having an inlet through which a reagent flows in and an outlet through which the reagent that has passed through the flow path flows out, The first electrode closest to the inlet is provided at a position away from the inlet by 2.0 times or more the width of the flow path.

FIGS. 1A to 1C are diagrams for explaining that a DNA probe and DNA to be examined are complementary. FIGS. 2A to 2D are diagrams illustrating a base sequence detection chip. FIGS. 3A and 3B are waveform diagrams showing an example of measurement results of oxidation current detected from the base sequence detection chip. Sectional drawing of the base sequence detection chip which performs an electric current detection using the three-electrode method. The figure which shows an example of the electric current detection system of a base sequence detection chip. The top view which shows the base sequence detection chip | tip by 1st Embodiment. The figure explaining the factor which a bubble is made. The figure which shows the relationship between a flow path width and the arrival position of a bubble. The top view which shows the base sequence detection chip | tip of 1st Embodiment in which the flow path was formed. The perspective view which shows the external shape of a base sequence detection apparatus. The perspective view which shows the structure of a base sequence detection apparatus. The top view of a base sequence detection apparatus. Sectional drawing of valve | bulb NOV. FIGS. 14A and 14B are diagrams illustrating the configuration of the valve NCV. The figure which shows the flow-path model of a base sequence detection apparatus. The figure which shows the amplification flow path of a base sequence detection apparatus. 17 (a) to 17 (e) are diagrams for explaining the operation of the base sequence detection device.

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

(Basic principle)
Before describing the base sequence detection chip of this embodiment, the basic principle of the base sequence detection chip will be described.

  This base sequence detection chip detects a base sequence, for example, a base sequence of DNA or RNA. DNA is composed of four bases of A (adenine), T (thymine), G (guanine), and C (cytosine), and is double-stranded. In the double-stranded form, the base A and the base T and the base G and the base C are combined in a specific combination. RNA is composed of four bases: A (adenine), U (uracil), G (guanine), and C (cytosine). In the following description, DNA detection will be described as an example, but RNA can be detected in the same manner.

  Since DNA fragments can be easily synthesized, the base sequence detection chip synthesizes DNA having a known sequence into a single strand, and fixes it on the substrate as a probe. The DNA to be examined is also made into a single strand and reacted with a DNA probe fixed on the substrate. If the DNA sequence to be inspected by the specimen is complementary to the DNA probe sequence, they are combined into a double strand. For example, as shown in FIG. 1 (a), when DNA probes are arranged in the order of TAGAC, if the DNA to be examined is arranged in the order of ATCTG (FIG. 1 (b)), the sequences are complementary to each other. Is. The combination of these single-stranded DNAs shown in FIG. 1 (c) into a double strand is called hybridization.

  As shown in FIG. 2A, the base sequence detection chip 500 is provided with a plurality of electrodes 520 spaced apart on a substrate 510 made of, for example, glass or silicon, and a DNA probe 530 having a different sequence is fixed on each electrode 520. The configuration is as follows. On this base sequence detection chip 500, a reagent containing a sequence of DNA to be examined is allowed to flow. If a DNA probe 530 having a sequence complementary to the sequence of the DNA to be inspected exists on the base sequence detection chip 500, the DNA 540 to be inspected reacts with the DNA probe 530 having a sequence complementary to the sequence of the DNA 540 to be inspected. Thus, hybridization occurs (FIGS. 2 (b) and 2 (c)). However, hybridization does not occur when the base sequence detection chip 500 does not have a DNA probe having a sequence complementary to the sequence of the DNA to be examined. The reagent is washed after flowing on the base sequence detection chip 500. Thereafter, a reagent (solution) containing the intercalating agent 550 is caused to flow on the base sequence detection chip 500. Then, the intercalating agent 550 binds to the double-stranded DNA probe 530 in which hybridization has occurred. When a voltage is applied to the base sequence detection chip 500 in this state, an oxidation current of the intercalating agent 550 flows through the electrode 520 on which the double-stranded DNA probe 530 in which hybridization has occurred is fixed (FIG. 2 (d)).

  An example of this oxidation current, that is, a signal from an electrode where hybridization has occurred is shown in FIG. 3A, and an example of a signal from an electrode where hybridization does not occur is shown in FIG. As can be seen from FIG. 3A, when the voltage applied to the base sequence detection chip 500 is increased, the oxidation current suddenly increases greatly at a voltage of about 500 mV. On the other hand, the signal value from the electrode where hybridization does not occur slightly increases when the voltage applied to the base sequence detection chip 500 is about 500 mV, but the degree of increase is higher than that shown in FIG. small. Thus, by determining which electrode the current is detected from, it is possible to know the DNA sequence to be examined.

(Current detection)
Next, an example of current detection of the base sequence detection chip 500 will be described with reference to FIG. 4 and FIG. This current detection is performed using, for example, a three-electrode method. In the base sequence detection chip 500 using this three-electrode method, for example, as shown in FIG. 4, a working electrode 520, a counter electrode 522, and a reference electrode 524 are provided on a substrate 510. The working electrode 520, the counter electrode 522, and the reference electrode 524 are provided apart from each other. At least one DNA probe 530 having the same arrangement (three in FIG. 4) is fixed to the working electrode 520 and connected to a terminal W provided on the substrate 510. For simplicity, only one working electrode 520 is shown, but a plurality of working electrodes are generally provided. The counter electrode 522 is connected to a terminal C provided on the substrate 510, and the reference electrode 524 is connected to a terminal R provided on the substrate 510.

  An example of the current detection system of the base sequence detection chip 500 configured as described above is shown in FIG. The current detection system 600 shown in FIG. 5 feeds back the voltage of the reference electrode 524 with respect to the input of the counter electrode 522, so that it can be desired in the solution regardless of variations in various conditions such as the electrode in the substrate 510 and the solution. It is a potentiostat which applies the voltage of.

  The potentiostat 600 changes the voltage of the counter electrode 522 so that the voltage of the reference electrode with respect to the working electrode 520 is set to a predetermined characteristic, and electrochemically measures the oxidation current caused by the intercalating agent.

  The working electrode 520 is an electrode to which the DNA probe 530 is fixed, and is an electrode for detecting a reaction current in the base sequence detection chip. The counter electrode 522 is an electrode that supplies a current into the base sequence detection chip by applying a predetermined voltage to the working electrode 520. The reference electrode 524 is an electrode that feeds back the voltage of the reference electrode 524 to the counter electrode 522 in order to control the voltage between the reference electrode 524 and the working electrode 520 so as to have a predetermined voltage characteristic. Thereby, the voltage by the counter electrode 522 is controlled, and it is possible to detect an oxidation current with high accuracy that is not affected by various detection conditions in the base sequence detection chip.

  A voltage pattern generation circuit 610 that generates a voltage pattern for detecting a current flowing between the electrodes is connected to the inverting input terminal of the inverting amplifier 612 for reference voltage control of the reference electrode 524 via the wiring 612b.

  The voltage pattern generation circuit 610 is a circuit that converts a digital signal input from a control mechanism of a base sequence detection control device (not shown) into an analog signal to generate a voltage pattern, and includes a DA converter.

Resistor _{R s} is connected to the wiring 612b. The inverting amplifier 612 has a non-inverting input terminal grounded and an output terminal connected to the wiring 602a. The wiring 612b on the inverting input terminal side of the inverting amplifier 612 and the wiring 602a on the output terminal side are connected by the wiring 612a. The wiring 612a is provided with a protection circuit 620 including a feedback resistor R _f ′ and a switch SW _f .

The wiring 602a is connected to the terminal C. The terminal C is connected to the counter electrode 522 on the base sequence detection chip 500. When a plurality of counter electrodes 522 are provided, a terminal C is connected in parallel to each. Thereby, a voltage can be simultaneously applied to a plurality of counter electrodes by one voltage pattern. The wiring 602a, the switch SW ₀ performing on-off control of voltage applied to the terminal C is provided.

  An excessive voltage is not applied to the counter electrode 522 by the protection circuit 620 provided in the inverting amplifier 612. Therefore, when a current is detected, an excessive voltage is applied to the counter electrode 522, and the solution is electrolyzed, so that the detection of the oxidation current of the desired intercalating agent is not affected, and stable measurement is performed. Is possible.

The terminal R is connected to the non-inverting input terminal of the voltage follower amplifier 613 by the wiring 603a. The inverting input terminal of the voltage follower amplifier 613 is short-circuited by the wiring 613b connected to the output terminal of the voltage follower amplifier 613 and the wiring 613a. The wiring 613b is resistor _{R f} is provided, the resistance _{R f} is connected to one end of the wiring 513b, are connected between the other end and the resistor _{R s,} the intersection of the wirings 612a and the wiring 612b. Thus, the voltage pattern generated by the voltage pattern generation circuit 610 is input to the voltage inverting amplifier 612 in which the voltage of the reference electrode 524 is fed back, and the voltage of the counter electrode 522 is changed based on the output obtained by inverting and amplifying such voltage. Control.

The terminal W is connected to the inverting input terminal of the trans-impedance amplifier 611 by the wiring 601a. The trans-impedance amplifier 611 has a non-inverting input terminal grounded, an output terminal connected to the wiring 611b, and connected to the inverting input terminal via the wiring 611a. Resistance _{R w} is provided in the 611a wiring. When the voltage at the terminal O on the output side of the trans-impedance amplifier 611 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 a control mechanism of a base sequence detection control device (not shown).

  Since there are a plurality of working electrodes 520, a plurality of terminals W and terminals O are provided corresponding to the working electrodes 520, respectively. Outputs from the plurality of terminals O are switched by a signal switching unit, which will be described later, and AD conversion is performed, whereby an electrochemical signal from each working electrode can be acquired as a digital value. The circuit such as the trans-impedance amplifier 611 between the terminal W and the terminal O may be shared by a plurality of working electrodes. In this case, a signal switching unit for switching wiring from the plurality of terminals W may be provided in the wiring 601a.

Using the potentiostat 600 having such a configuration, the current measurement of the base sequence detection chip is performed as follows.
1. First, the potential of the working electrode 520 is made constant with respect to the potential of the reference electrode 524 using the potentiostat 600.
2. The working electrode 520 electrolyzes the intercalating agent on this electrode 520.
3. The current required to maintain electrolysis at the working electrode 520 is the current Ic flowing from the counter electrode 522.
4). At this time, the potentiostat 600 accurately measures the current flowing between the working electrode 520 and the counter electrode 522. That is, almost no current flows through the reference electrode 524.

(First embodiment)
Next, the base sequence detection chip 500 according to the first embodiment is shown in FIG. The base sequence detection chip 500 of this embodiment includes 40 working electrode groups 520 _{1 to} 520 ₄₀ , counter electrodes 522 a and 522 b, and a reference electrode 524 on a solid phase substrate 510. Each of the counter electrodes 522a and 522b is indicated as CE on the drawing, and the reference electrode 524 is indicated as RE. The solid phase substrate 510 is formed from, for example, a glass substrate or a silicon substrate.

Forty working electrode groups 520 _{1 to} 520 ₄₀ are arranged by being divided into first to fourth portions. The first part is composed of 10 sets of working electrode groups 520 _{1 to} 520 ₁₀ and is arranged from left to right on the drawing. The second portion includes 10 sets of working electrode groups 520 _{11 to} 520 ₂₀ , which are arranged below the first portion on the drawing and arranged from right to left on the drawing. The third portion includes 10 sets of working electrode groups 520 _{21 to} 520 ₃₀ , which are arranged below the second portion on the drawing and arranged from left to right on the drawing. The fourth portion includes 10 sets of working electrode groups 520 _{31 to} 520 ₄₀ , which are arranged below the third portion on the drawing and arranged from right to left on the drawing.

Each working electrode group 520 _i (i = 1,..., 40) includes three working electrodes that are spaced apart from each other, and are numbered 3i-2, 3i-1, and 3i, respectively. Yes. For example, the working electrode group 520 ₁ is provided with a working electrode 1, 2, 3 number of assigned working electrode group _{520 10} is provided with a working electrode number of 28, 29 and 30 attached. DNA probes having the same DNA sequence are fixed to the three working electrodes of each working electrode group 520 _i (i = 1,..., 40). Each working electrode is made of gold, for example.

Counter electrode 522a has a U-shape, one end of the U-shape are disposed proximate to the working electrode group 520 ₁₀ of the first portion, the other end of the U-shaped operation of the second portion The electrode group 520 ₁₁ is disposed in the vicinity. Counter electrode 522b has a U-shape, one end of the U-shape is disposed proximate to the working electrode group 520 ₃₀ of the third portion, the other end of the U-shaped operation of the fourth portion The electrode group 520 ₃₁ is disposed in the vicinity.

The reference electrode 524 has a U-shape, is arranged at one end of the U-shaped proximate to the working electrode group 520 ₂₀ of the second portion, the other end of the U-shaped operation of the third portion The electrode group 520 ₂₁ is disposed in the vicinity.

In addition, the substrate 510 is provided with terminals W that are electrically connected to the working electrodes of each working electrode group 520 _i (i = 1,..., 40). The terminals W are assigned numbers 3i-2, 3i-1, 3i corresponding to the working electrodes of each working electrode group 520 _i (i = 1,..., 40). For example, each of the terminal W of the numbers 1, 2, and electrically connected to the working electrode of the working electrode group 520 ₁ number 1, 2, 3, respectively terminal W of numbers 118, 119, 120, a working electrode Electrically connected to working electrodes of group 520 ₄₀ , number 118, 119, 120;

The substrate 510 is further provided with C terminals 523a and 523b, R terminals 525a and 525b, an X terminal 526, Y terminals 528a and 528b, and a plurality of conduction detection terminals 58. The C terminal 523a is electrically connected to the counter electrode 522a, and the C terminal 523b is electrically connected to the counter electrode 522b. The R terminals 525a and 525b are connected to the reference electrode 524, respectively. The X terminals 526a and 526b are terminals provided at both ends of the base sequence detection chip 500, and a voltage is applied between the terminals 526a and 526b to detect whether or not the base sequence detection chip 500 is conductive. Similarly, Y terminals 528a and 528b are provided at both ends of the base sequence detection chip 500, and a voltage is applied between these terminals 528a and 528b to detect whether or not the base sequence detection chip 500 is conductive. It is. The plurality of conduction detection terminals 580 are provided corresponding to the working electrodes of the working electrode groups 520 _{1 to} 520 ₄₀ , and each conduction detection terminal 580 is connected to the corresponding working electrode and electrically connected to the corresponding working electrode. By applying a voltage between the terminal W to be connected, it is detected whether or not the corresponding working electrode and the terminal W are electrically connected. As shown in FIG. 6, the terminals are divided into upper and lower groups on the drawing so as to sandwich the working electrode group 520 _i (i = 1,..., 40).

The base sequence detection chip 500 is attached to a base sequence detection device described later. When mounted, to flow reagents containing intercalating agent toward the working electrode 520 ₁₀ from the working electrode group 520 _1, the flow path is formed on the first portion. Further, also on the counter electrode 522a, the flow path to flow is the reagent toward the working electrode _{520 11} from the working electrode group _{520 10} is formed. As flows the reagent toward the working electrode 520 ₂₀ from the working electrode group 520 _11, the flow path is formed on the second portion. A flow path is also formed on the reference electrode 524 so that the reagent flows from the working electrode group 520 ₂₀ toward the working electrode 520 ₂₁ . A flow path is formed on the third portion so that the reagent flows from the working electrode group 520 ₂₁ toward the working electrode 520 ₃₀ . Further, also on the counter electrode 522b, the flow path to flow is the reagent toward the working electrode _{520 31} from the working electrode group _{520 30} is formed. A flow path is formed on the fourth portion so that the reagent flows from the working electrode group 520 ₃₁ toward the working electrode 520 ₄₀ . These flow paths are formed by the region of the base sequence detection chip 500 in which the first to fourth parts of the working electrode group, the counter electrode, and the reference electrode are formed, and an upper cover that covers these regions. As the upper lid, for example, a packing of a base sequence detection device is used.

The reagent including the intercalating agent includes a flow path on the first portion, a flow path on the counter electrode 522a, a flow path on the second portion, a flow path on the reference electrode 524, a flow path on the third portion, It passes through the flow path on the counter electrode 522b and the flow path on the fourth portion in this order. In FIG. 6, the three working electrodes of each working electrode group 520 _i (i = 1,..., 40) are arranged along the direction in which the reagent flows.

  The inventors of the present invention have made extensive studies on the cause of bubbles generated in the flow path on the electrode of the base sequence detection chip 500 formed by the upper lid. As a result, the following was found. As shown in FIG. 7, the entrance of the base sequence detection chip 500 into which the reagent flows is configured to be provided on the upper lid 770 substantially perpendicular to the base sequence detection chip 500. Therefore, the reagent flows from the inlet 760a vertically downward toward the base sequence detection chip 500 as indicated by an arrow 765a. For this reason, a stagnation 790 is generated in the region of the flow path 780 immediately below the inlet, and bubbles are generated by this stagnation, and the bubbles reach the region on the electrode 520 through the flow path 780 as indicated by an arrow 765b.

  Therefore, the present inventors conducted an experiment and investigated how far the bubble was sent in the flow path from the region directly under the inlet. The result is shown in FIG. As can be seen from FIG. 8, when the channel width was 1 mm, the arrival position of the bubbles was 1.6 times the channel width, and when the channel width was 1.2 mm, it was 1.64 times. From the above, it has been found that bubbles are not sent to a region separated by 2.0 times or more the width of the flow path 780.

  Based on this result, in the base sequence detection chip 500 of the first embodiment, the electrode closest to the inlet into which the reagent flows is arranged at a distance of 2.0 times or more the width of the channel 780 from the inlet 760a. That is, the distance Lof from the inlet 760a to the nearest electrode is 2.0 times or more the width of the flow path 780. From the above experimental results, it was found that the distance Lof is preferably 2 to 3 times the width of the flow path 780.

  FIG. 9 shows the base sequence detection chip 500 in which the flow path 780 is formed by the upper lid 770. The distance Lof from the inlet 760a into which the reagent flows into the working electrode 520a in the channel 780 closest to the inlet 760a is 2.0 times or more the width Lw of the channel 780. In the present embodiment, the outlet 760b side from which the reagent flows out from the base sequence detection chip 500 is arranged in the same manner as the inlet side. That is, the working electrode 520b closest to the outlet 760b is disposed at a distance of 2.0 times or more the width Lw of the flow path 780 from the outlet 760b.

  As described above, according to the present embodiment, it is possible to provide a base sequence detection chip capable of suppressing air bubbles from being sent to the channel on the electrode.

(Base sequence detection device equipped with the base sequence detection chip of the first embodiment)
Next, an example of a base sequence detection device that mounts the base sequence detection chip of the first embodiment and detects the DNA sequence of a specimen will be described. An outline drawing of the base sequence detection apparatus is shown in FIG. As shown in FIG. 11, the base sequence detection apparatus 700 includes a lower plate 710, a packing 720, an upper plate 730, a cover 740, and a cap 750. The packing 720 is inserted between the lower plate 710 and the upper plate 730. Cover 740 is attached to upper plate 730. Cap 750 is attached to cover 740 by a hinge. The lower plate 710, the upper plate 730, the cover 740, and the cap 750 are made of, for example, transparent polycarbonate (PC), and the packing 720 is made of, for example, a transparent elastomer.

  The lower plate 710 is provided with a bank 710a along the periphery, and the inner surface of the bank 710a is lower than the bank 710a. An opening 710b is provided inside the bank 710a. Around the opening 710b, a base sequence detection chip 500 is placed and a support portion 710b1 for supporting the base sequence detection chip 500 is provided. The support portion 710b1 is provided on the bottom side of the opening 710b so that the upper surface of the base sequence detection chip 500 is lower than the inner surface of the bank 710a when the base sequence detection chip 500 is placed on the support portion 710b1. It is done. In addition, the packing 720 is arrange | positioned so that the area | region inside the bank 710a may be covered.

  FIG. 12 shows a top view of the base sequence detection device 700 when the base sequence detection chip 500 is mounted. On the inner side of the bank 710a of the lower plate 710, four depressions 710c1, 710c2, 710c3, and 710c4 that form the syringe portion 710c are provided. As will be described later, the recess 710c1 forms a tank together with the expansion of the packing 720, and a liquid containing a sample DNA, for example, a sample is stored in the tank. The recess 710c2 forms a tank together with the expansion of the packing 720, and for example, cleaning liquid is stored in the tank. For example, a reagent containing an intercalating agent is stored in the recess 710c3. The recess 710c4 forms a tank together with the expansion of the packing 720, and for example, cleaning liquid is stored in the tank. Injection portions 710d1, 710d2, 710d3, and 710d4 for injecting liquid are provided on the respective inlet sides of the recesses 710c1, 710c2, 710c3, and 710c4. 710 c 2, 710 c 3, and 710 c 4 are connected to each other through a groove serving as a flow path provided in the lower plate 710.

  An air vent 710e1 is provided on the entrance side of the recess 710c1, an air vent 710e2 is provided on the exit side of the recess 710c2, an air vent 710e3 is provided on the exit side of the recess 710c3, and an air vent 710e4 is provided on the exit side of the recess 710c4. It has been.

  Further, on the inner side of the bank 710a of the lower plate 710, a normally closed valve NCV provided on the outlet side of each of the recesses 710c1, 710c2, 710c3, 710c4, an amplification channel 710f for amplifying DNA in the sample, A normally open valve NOV provided at the inlet and outlet of the amplification flow path 710f, and recesses 710g1 and 710g2 which become waste liquid tanks 711g1 and 711g2 are provided to keep the waste liquid in the base sequence detection device. . The waste liquid tanks 711 g 1 and 711 g 2 are formed by the depressions 710 g 1 and 710 g 2, the packing 720, and the upper plate 730.

  The valve NCV is formed by the lower plate 710, the packing 720, and the cover 740, and the valve NOV is formed by the lower plate 710 and the packing 720. The valve NCV is used for storing a reagent or the like in the syringe unit 710c. The valve NOV is used to close the flow path so that a reagent or the like does not move during DNA amplification.

  Further, the valve NCV provided on the outlet side of the recesses 710c1 and 710c2 is connected to the valve NOV provided on the inlet side of the amplification flow path 710f through the flow path formed in the lower plate 710. The valve NCV provided on the outlet side of the recesses 710c3 and 710c4 is connected to the inlet of the channel on the base sequence detection chip 500 formed by the packing 720 and the channel formed on the lower plate 710. A valve NOV provided on the outlet side of the amplification flow path 710f is connected to the flow path formed on the base sequence detection chip 500 formed by the packing 720 and the flow path formed on the lower plate 710. The outlet of the channel on the base sequence detection chip 500 formed by the packing 720 is connected to the recess 710g1 by the channel formed in the lower plate 710.

  As shown in FIG. 11, the packing 720 is provided with a dome-like bulge convex toward the upper plate 730 in each of the regions corresponding to the dents 710c1, 710c2, 710c3, 710c4, and the dent 710c1 corresponding to this bulge. , 710c2, 710c3, and 710c4 form a tank of the syringe unit 710c. Further, the packing 720 has a tube structure in cross section in a region where the valve NCV and the valve NOV are formed as described later, and a flow path is formed by the tube structure and the lower plate 710. The region of the lower plate 710 corresponding to this tube structure is flat without grooves. Further, the surface of the packing 720 covering the channel groove formed in the lower plate is flat. A flow path is formed by the flat surface of the packing and the groove.

  Further, the packing 720 is provided with an opening in a region corresponding to the depressions 710g1 and 710g2, and the terminals of the DNA chip 500 (for example, the terminal W connected to the working electrode 520 and the terminal C connected to the counter electrode 522 shown in FIG. 6). , An opening is provided corresponding to a region where a terminal R connected to the reference electrode 524 is provided. Therefore, when the base sequence detection chip 500 to be mounted is the chip shown in FIG. 6, two openings are required.

  The upper plate 730 is provided with a first opening in a region corresponding to the swelling of the packing 720. Through this first opening, a rod extends from a DNA sequence detection control device (not shown) and pushes the upper surface of the tank, that is, the upper surface of the bulge of the packing 720, whereby the liquid is discharged from the tank to the flow path. At this time, the valve NCV provided on the outlet side of the tank is opened. Further, the upper plate 730 is provided with a second opening corresponding to the region where the terminal of the base sequence detection chip 500 is provided. In the region where the first opening is provided, the position of the upper surface is lower than the region where the second opening is provided. A cover 740 is attached to the region where the first opening is provided. Further, in the region where the first opening is provided, an opening is provided in a region corresponding to the valve NCV.

  FIG. 13 shows a cross section of the valve NOV cut along a plane orthogonal to the direction in which the flow path extends. As shown in FIG. 13, the valve NOV has a tube structure 720a, and the tube structure 720a and the lower plate 710 serve as a valve that is normally open. The valve NOV closes the flow path by crushing the tube structure of the packing 720 with a rod 810 from a DNA sequence detection control device (not shown). In addition, an opening into which the rod 810 is inserted is provided in a region of the upper plate 730 corresponding to a region where the valve NOV is provided.

  As shown in FIG. 14A, on the back side of the cover 740, a rod 740a for crushing the tube structure of the packing 720 is provided at the tip of the cantilever corresponding to the position where the valve NCV is provided. The valve NCV is normally closed by the rod 740a. As shown in FIG. 14B, the valve NCV is opened when the rod 820 from the base sequence detection control apparatus lifts the cantilever, and the valve NCV is opened, and the flow path having the tube structure of the packing 720 is formed. Opens. Note that an opening into which the rod 820 is inserted is provided in a region of the lower plate 710 corresponding to a region in which the valve NCV is provided.

  FIG. 15 shows a flow channel model of the base sequence detection device 700. The liquid containing the sample DNA is injected from the inlet 710d1 and stored in the tank 711d1, and the cleaning liquid is injected from the inlet 710d2 and stored in the tank 711d2. The reagent containing the intercalating agent is injected from the inlet 710d3 and stored in the tank 711d3, and the cleaning liquid is injected from the inlet 710d4 and stored in the tank 711d4. Air ports 711 e 1, 711 e 2, 711 e 3, and 711 e 4 are provided for extracting air stored in the tanks 711 d 1, 711 d 2, 711 d 3, and 711 d 4. The reagent from the tank 711d3 or the cleaning liquid from the tank 711d3 flows into the inlet of the channel 712 formed on the base sequence detection chip 500 through the channel. At this time, in order to prevent the reagent or the cleaning liquid from leaking out from the packing 720, the flow path is once lifted upward in the vicinity of the inlet of the flow path 712 to be in a horizontal state and then lowered to the inlet of the flow path 712. Connecting. This is because, in the region where the flow path is in a horizontal state, as can be seen from FIG. 11, the lower plate is flat, and in this region, the packing 720 has a tube structure. The waste liquid tanks 711g1 and 711g2 are connected by a flow path, and the waste liquid moves to the waste liquid tank 711g2 when the tank 711g1 is nearly full.

  As shown in FIG. 16, the amplification channel 710f is provided with a plurality of wells 710f1 in which the liquid is temporarily stored at equal intervals in the channel. The inventors of the present application have found that the amplification channel 710f having such a structure can perform multi-amplification capable of amplifying a plurality of different DNAs. In addition, it is preferable that the space | interval between adjacent wells 710f is 4 mm or more along a flow path.

  Next, the operation of the base sequence detection apparatus will be described with reference to FIGS. 17 (a) to 17 (e). 17 (a) to 17 (e) are plan views of the flow channel model shown in FIG.

  First, the liquid (sample) containing the sample-extracted DNA, the first washing liquid, the reagent containing the intercalating agent, and the second washing liquid are injected into the tanks 711d1, 711d2, 711d3, and 711d4 of the base sequence detection device 700, respectively. . This base sequence detection device 700 is mounted on a base sequence detection control device (not shown) (FIG. 17A).

  Subsequently, the valve NCV on the outlet side of the tank 711d1 holding the sample in the base sequence detection device 700 is opened by the control mechanism of the base sequence detection control device. Thereafter, the sample is sent to the amplification flow path 710f by crushing the tank 711d1 holding the sample with the rod of the base sequence detection control device. After the sample is sent to the amplification channel 710f, the valve NOV at the entrance / exit of the amplification channel 710f is closed, and the amplification channel 710f is heated by the heating mechanism (eg, heater) of the base sequence detection control device. Then, an amplification reaction is caused in the DNA in the sample (FIG. 17B).

  Next, the valve NCV on the outlet side of the tank 711d2 in which the first cleaning liquid is stored is opened and the valve NOV on the inlet / outlet of the amplification channel 710f is opened by the rod of the base sequence detection controller. Subsequently, the tank 711d2 is crushed by the rod of the base sequence detection control device to send the first cleaning liquid to the amplification flow path 710f. Then, the sample containing the DNA in which the amplification reaction has occurred is pushed out by the first washing solution and flows into the channel 712 formed on the base sequence detection chip. After the sample is sent onto the base sequence detection chip, the valve NOV at the entrance / exit of the amplification flow path 710f is closed. Here, the base sequence detection chip is heated by a heating / cooling mechanism (for example, a Peltier element) of the base sequence detection control device. When the DNA in the sample has a DNA sequence complementary to the DNA probe sequence of the base sequence detection chip, a hybridization reaction occurs in the channel 712 (FIG. 17 (c)).

  Next, the valve NCV on the outlet side of the tank 711d4 in which the second cleaning liquid is stored is opened by the control mechanism of the base sequence detection control device. Subsequently, the second cleaning liquid is sent to the flow path 712 by crushing the tank 711d4 with the rod of the base sequence detection control device. At this time, the base sequence detection chip is heated by the base sequence detection controller. Thereby, non-specific substances adsorbed on the DNA probe of the base sequence detection chip are washed away. The washed away non-specific material is sent to the waste liquid tank 711g1 (FIG. 17 (d)).

  Next, the valve NCV on the outlet side of the tank 711d3 in which the reagent containing the intercalating agent is stored is opened by the control mechanism of the base sequence detection control device. Subsequently, the reagent is sent to the channel 712 by crushing the tank 711d3 with the rod of the base sequence detection control device. At this time, the base sequence detection chip and the channel 712 are held at room temperature (for example, 25 ° C.) by the heating / cooling mechanism of the base sequence detection control device.

  Next, the base sequence detection control device uses a base sequence detection chip terminal (for example, a terminal W connected to the working electrode 520 shown in FIG. 6, a terminal C connected to the counter electrode 522, a terminal R connected to the reference electrode 524, etc.). A DNA sequence in the sample is detected by applying a voltage to and detecting the current from each working electrode.

  As described above, it is possible to provide a base sequence detection chip capable of suppressing air bubbles from being sent to the channel on the electrode. In addition, the number of detectable genes can be increased. Moreover, since the expensive reagent is held in the tank by the amount necessary for the detection process, the DNA sequence can be detected at a low cost.

  Although several embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the scope of the invention. These embodiments and modifications thereof are included in the invention described in the claims and equivalents thereof as well as included in the scope and gist of the invention.

500 base sequence detection chip 510 solid phase substrate 520 electrode (working electrode)
520a Working electrode closest to the inlet 520b Working electrode closest to the outlet 522 Counter electrode 524 Reference electrode 530 DNA probe 540 DNA to be examined
550 Insertion agent 600 Potentiostat 760a Channel inlet 760b Channel outlet 770 Upper lid 780 Channel C terminal R terminal W terminal

Claims (8)

A plurality of electrode groups arranged apart from each other on the substrate, each electrode group having a plurality of first electrodes arranged apart from each other;
A probe provided corresponding to each first electrode, fixed to the corresponding first electrode, and having a base sequence;
A cover member that is provided on the substrate so as to cover the plurality of electrode groups along the arrangement direction of the plurality of electrode groups and forms a flow path with the substrate, the inlet through which a reagent flows, the flow A cover member having an outlet through which the reagent that has passed through the passage flows out;
With
The base sequence detection chip, wherein the first electrode closest to the inlet is provided at a position separated from the inlet by 2.0 times or more the width of the flow path.   The base sequence detection chip according to claim 1, wherein the first electrode closest to the inlet is located at a distance of 2 to 3 times the width of the flow path from the inlet.   The outlet is provided so that the reagent flows out substantially perpendicularly to the substrate, and the first electrode closest to the outlet is provided at a position separated from the outlet by 1.4 times or more of the width of the channel. The base sequence detection chip according to claim 1 or 2, wherein:   The base sequence detection chip according to any one of claims 1 to 3, wherein probes having the same base sequence are fixed to the plurality of first electrodes in each electrode group.   5. The base sequence detection chip according to claim 1, wherein probes having different base sequences for each of the electrode groups are fixed to the plurality of first electrodes. A second electrode provided on the substrate, the second electrode for supplying a current to the substrate by applying a voltage between the second electrode and the plurality of first electrodes;
A third electrode provided on the substrate, the third electrode for controlling the voltage between the third electrode and the plurality of first electrodes to have a predetermined voltage characteristic;
The base sequence detection chip according to any one of claims 1 to 5, further comprising: The plurality of electrode groups are divided into first to fourth portions, the second portion is disposed between the first portion and the fourth portion, and the third portion is the second portion and the second portion. Between the four parts,
The second electrode is divided into two parts;
One portion of the second electrode has a U-shape, one end of the second electrode is provided with one end close to one end of the first portion, and the other end of the first electrode. Provided close to one end of the second part on the same side as the one end of the part;
The other part of the second electrode has a U shape, and the other part of the second electrode has one end provided close to one end of the third part and the other end of the third electrode. Provided close to one end of the fourth portion on the same side as the one end of the portion;
The third electrode has a U-shape, and one end of the third electrode is provided close to the other end of the second part, and the other end is close to the other end of the fourth part. Provided,
The upper lid is provided so as to cover the first and fourth portions and further cover the second and third electrodes along the respective shapes of the second and third electrodes,
The upper lid is formed on the first portion, the one portion of the second electrode, the second portion, the third electrode, the third portion, the other portion of the second electrode, and the fourth portion. The base sequence detection chip according to claim 6, which is continuous. A plurality of first terminals provided on the substrate and respectively connected to the plurality of first electrodes in the plurality of electrode groups;
A second terminal provided on the substrate and connected to the second electrode;
A third terminal provided on the substrate and connected to the third electrode;
The base sequence detection chip according to claim 6 or 7, further comprising:

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