JP6953501B2 - Nucleic acid detection cassette - Google Patents

Description

Embodiments relate to nucleic acid detection cassettes used in nucleic acid testing devices that detect nucleic acids using electrical signals.

In recent years, with the development of genetic engineering, in the medical field, it is becoming possible to diagnose or prevent diseases by genes. This is called genetic diagnosis, which allows the diagnosis or prediction of a disease before the onset of the disease or at an extremely early stage by detecting a human genetic defect or genetic change that causes the disease. It becomes possible to do. In addition, along with the decoding of the human genome, research on the relationship between genotypes and epidemics is progressing, and treatments tailored to each individual's genotype (tailor-made medical treatment) are becoming a reality. Therefore, it is very important to easily detect genes and determine genotypes.

As a nucleic acid test device, a device using a DNA chip is known (Patent Document 1). The DNA chip has a detection region composed of a plurality of sensor units on which a nucleic acid probe is immobilized on a substrate, and has a feature that a large number of nucleic acid sequences can be detected at one time. Generally, the nucleic acid probe is immobilized on the sensor surface by being dropped onto each sensor unit in a state of being dissolved in a liquid. Further, in order to immobilize different nucleic acid probes for each sensor unit, it is said that it is necessary to avoid contact between the sensor units.

On the other hand, a cassette called μ-TAS, which can sequentially perform a plurality of reactions involving a plurality of reagents in one cassette, has been actively researched and developed. The μ-TAS is composed of a reagent holding region, a nucleic acid reaction region, a detection region, and the like, and has a flow path connecting them.

Further, a nucleic acid test device for detecting a nucleic acid using a nucleic acid detection cassette containing the DNA chip of the current detection method has also been developed. When nucleic acid detection is performed using such a cassette, it is necessary to use a plurality of reagents and perform a plurality of treatments and reactions, such as extraction, purification, nucleic acid amplification reaction, and nucleic acid hybridization reaction, under predetermined conditions. be. Since these reagents are generally expensive, it is desired to reduce the amount used as much as possible and obtain a lot of information from such a small sample solution. Therefore, it is desired to realize a nucleic acid detection cassette capable of efficiently supplying a sample solution, a cleaning solution, and a reagent to each sensor unit by arranging each sensor unit in the flow path formed in the cassette. ..

When supplying the sample solution, cleaning liquid, and reagent, it is conceivable to send air from the outside into the flow path by a pump, but it is difficult to send the sample solution to the detection unit with high accuracy. ing.

Japanese Unexamined Patent Publication No. 2014-60925 JP-A-2009-109334 Japanese Unexamined Patent Publication No. 2008-241397 WO2008 / 087828

Patent Document 1 discloses a structure in which a nucleic acid detection cassette includes a sample syringe, a cleaning syringe, and an insertion agent syringe, and a sample solution (sample), a cleaning solution, and a reagent (insertion agent solution) flow into the sensor unit from these syringes. ing. The nucleic acid detection cassette disclosed in Patent Document 1 does not have an amplification unit, and an amplified sample solution (sample) is prepared and filled in a sample syringe. A nucleic acid detection cassette having such a structure is required to be applicable to a nucleic acid test apparatus capable of automating from sample amplification to detection of sample electrochemical reaction.

Patent Document 2 discloses a structure in which a microchemical chip includes a pump chamber, and the pump chamber is operated by a microchip actuator. In this microchemical chip, the driving liquid is pushed out from the driving liquid storage unit, the sample is transferred to the amplification unit, and the sample is transferred to the detection unit. In this structure, since the microchemical chip includes a pump chamber and a drive liquid storage portion, there is a possibility that the request for miniaturization of the microchemical chip cannot be met.

Patent Document 3 discloses a DNA chip processing apparatus that feeds a flushing liquid, a cleaning liquid, and a staining liquid by a liquid feeding pump. In this processing device, a liquid feeding mechanism is independently provided outside the DNA chip. Therefore, the DNA chip processing apparatus cannot respond to the request for cassette formation containing the DNA chip, and cannot respond to the request for the realization of an automated nucleic acid testing apparatus.

Patent Document 4 discloses a microchip having a main flow path and a sub flow path. A driving liquid is supplied to the microchip by a micropump in order to send a sample and a reagent. In such a structure, the microchip for supplying the driving liquid requires a plurality of openings, the reagent or the sample may leak to the outside, and the microchip is completely hermetically sealed. There is a risk that it will not be possible to realize automated nucleic acid testing on the chip.

In any of the patent documents, there is a demand for a nucleic acid test device that cannot accurately send a sample solution to a detection unit and can automate the process from sample amplification to detection of a sample electrochemical reaction.

As described above, in order to reduce the price of the nucleic acid detection cassette, it is required to reduce the size of the nucleic acid detection cassette, and in order to reduce the size of the nucleic acid detection cassette, it is necessary to highly integrate each member. Further, it is required that the nucleic acid detection cassette can be used for a nucleic acid test device that automates the process from sample amplification to detection of the electrochemical reaction of the sample.

An object of the present invention is to provide a nucleic acid detection cassette that is highly integrated and can be used in a nucleic acid test apparatus that automates the process from sample amplification to detection of sample electrochemical reaction.

In order to solve the above problems, according to the embodiment,
The first, second, which are formed to be deformable from the outside by a first plate made of a hard material and a packing made of an elastic material, and store a sample solution, a first cleaning liquid, an insertion agent liquid, and a second cleaning liquid , respectively. With the 3rd and 4th syringes,
An injection unit capable of injecting a sample solution into the first syringe from the outside,
An amplification flow path having an input side and an output side is provided, and the amplification flow path is an amplification portion formed by the first plate and the packing, and the amplification flow path has a plurality of wells. Displaced at intervals, different types of primer sets are immobilized in the plurality of wells, and the nucleic acids in the sample solution are released by releasing the primer set into the sample solution supplied into the amplification flow path. And the amplification part where the amplification product is generated by amplification
Fifth and sixth valves provided on the input side and the output side of the amplification flow path, respectively, the first and first valves that confine the sample solution in the amplification section during the nucleic acid amplification in the amplification section. The second valve and
It has a plurality of electrodes and a plurality of electrode pads connected to the plurality of electrodes, which are placed on the first plate, and the plurality of electrodes electrically perform a hybridization reaction corresponding to the amplification product. A chip containing a working electrode to which a plurality of types of nucleic acid probes are fixed for detection is formed between the chip and the packing, and the plurality of electrodes are arranged and have an input side and an output side. A detection unit provided with a detection flow path, the sample solution containing the amplification product is supplied from the amplification unit, and the detection unit detects the amplification product, and
The first, second, third, fourth, and fifth flow paths formed between the first plate and the packing.
The first and second channels are communicated with the first and second syringes, respectively, so that the sample solution is supplied and then the first cleaning solution is supplied to the amplification section. It is connected in common with the first valve on the input side of the amplification flow path.
The fifth flow path is connected to the second valve on the output side of the amplification flow path so that the sample solution containing the amplification product is supplied from the amplification section to the detection section in the supply of the first cleaning solution. By connecting to the input side of the detection flow path ,
The first, second, and third channels connecting the third and fourth flow paths to the input side of the detection flow path so as to supply the insertion agent solution and the second cleaning solution that do not pass through the amplification unit, respectively. , 4th and 5th channels,
Equipped with
A second plate made of a hard material is provided on the packing, and the packing is pressed and sandwiched between the first plate and the second plate, and the first, second, and second plates are inserted. The 3rd and 4th syringes, the amplification unit, the detection unit , the 1st, 2nd, 3rd, 4th and 5th flow paths and the 1st and 2nd valves are formed by being liquidtightly sealed. the nucleic acid detection cassette is provided with a not that structure is.

It is a block diagram which shows schematic the nucleic acid test apparatus which concerns on embodiment. It is a perspective view which shows the appearance of the nucleic acid detection cassette mounted on the nucleic acid test apparatus shown in FIG. It is a decomposition perspective view which shows roughly by disassembling the nucleic acid detection cassette shown in FIG. It is a perspective plan view which shows the internal structure schematicly by seeing through the nucleic acid detection cassette shown in FIG. FIG. 5 is a plan view schematically showing a flow path provided in the nucleic acid detection cassette shown in FIG. 2 and each part connected to the flow path. It is a perspective view which shows typically the flow path provided in the nucleic acid detection cassette shown in FIG. 2 and each part connected to the flow path. It is a top view which shows roughly the form of the amplification part of the nucleic acid detection cassette shown in FIG. It is a top view which shows the planar structure of the DNA chip of the nucleic acid detection cassette shown in FIG. 2 schematically. It is a perspective view which shows the appearance which removed the cap and the cover from the nucleic acid detection cassette shown in FIG. It is a partial cross-sectional view schematically showing the form of the normally open valve provided in the nucleic acid detection cassette shown in FIG. FIG. 10 is a partial cross-sectional perspective view schematically showing a normally open valve and a part of a valve rod located on the normally open valve, which is shown in FIG. It is a perspective view which shows the form of the back surface of the packing shown in FIG. 3 schematically. It is a perspective view from the back side which shows roughly the assembly position of the upper plate and packing of the nucleic acid test cassette shown in FIG. It is a perspective view which shows typically the structure which attached the packing to the back surface of the upper plate shown in FIG. FIG. 3 is a perspective view schematically showing a process of attaching an upper plate to which the packing shown in FIG. 14 is attached to a lower plate. FIG. 1 is a side view schematically showing the internal structure of the nucleic acid testing apparatus shown in FIG. FIG. 1 is a flowchart showing a process of electrochemically inspecting a sample in a nucleic acid inspection apparatus. FIG. 5 is a plan view schematically showing the supply of a sample to the amplification unit in the nucleic acid detection cassette shown in FIG. FIG. 5 is a plan view schematically showing the supply of the first washing liquid to the amplification section in the nucleic acid detection cassette shown in FIG. 6 and the supply of a sample to the detection section. It is a top view which shows typically the supply of the 2nd washing liquid to the detection part in the nucleic acid detection cassette shown in FIG. It is a top view which shows typically the supply of the insertion agent liquid to the detection part in the nucleic acid detection cassette shown in FIG.

Hereinafter, the nucleic acid detection cassette according to the embodiment will be described in detail with reference to the drawings.

(Rough configuration of nucleic acid test device)
FIG. 1 shows a block of a nucleic acid test device 8 according to an embodiment. The nucleic acid test device 8 is configured to be capable of automating from the amplification of the sample to the detection of the electrochemical reaction of the sample. The nucleic acid test device 8 physically controls a liquid-tightly sealed nucleic acid detection cassette 10, a measuring unit 12 electrically connected to the nucleic acid detection cassette 10, and a flow path system provided in the nucleic acid detection cassette 10 from the outside. It is provided with a liquid feed control mechanism 16 for driving and controlling the nucleic acid, a temperature control mechanism 14 for controlling the temperature of each part of the nucleic acid detection cassette 10, and the like. The nucleic acid detection cassette 10 contains a DNA chip 6 as described in detail later. As will be described in detail later, the DNA chip 6 is provided with a detection flow path through which a solution such as a sample flows. A plurality of electrodes for extracting a hybridization signal are arranged in the detection flow path on the DNA chip 6. These electrodes include a working electrode, a counter electrode and a reference electrode on which a nucleic acid probe containing a sequence complementary to the base sequence to be detected is immobilized. At least one counter electrode and a reference electrode are provided so as to face the working electrode. The measuring unit 12 shown in FIG. 1 is a three-electrode system in which the measuring unit 12 is connected to the DNA chip 6 and feeds back (negative feedback) the voltage of the reference electrode in response to the application of the input voltage between the working electrode and the counter electrode in the DNA chip 6. It is configured as a Potencio Stat. Therefore, according to the measuring unit 12, a desired voltage can be applied to the solution without changing the electrodes in the cell of the nucleic acid detection cassette 10 or various conditions such as the solution, and the electrochemical reaction can be performed. (Hereinafter referred to as "electrochemical current") can be measured electrochemically.

The measurement unit 12, the temperature control mechanism 14, and the liquid feed control mechanism 16 described above are connected to the device control unit 18, and the device control unit 18 is connected to the computer 4 outside the nucleic acid test device 8 via an interface (not shown). It is connected. In response to a command such as an operation instruction from the computer 4, the device control unit 18 controls the measurement unit 12, the temperature control mechanism 14, and the liquid feed control mechanism 16 according to the built-in program. The apparatus control unit 18 controls the liquid feed control mechanism 16 to feed a solution such as a sample, and controls the temperature control mechanism 14 to control the temperature of the sample solution to perform a nucleic acid amplification reaction, a hybridization reaction, and a hybrid reaction. It is causing an electrochemical reaction. Further, the device control unit 18 controls the measurement unit 12 to detect the electrochemical reaction following the hybridization reaction, and transfers the obtained detection signal to the computer 4 as detection data. Computer 4 analyzes the transferred detection data to identify the presence of nucleic acids in the sample solution. The nucleic acid test device 8 shown in FIG. 1 automates from amplification of nucleic acid in the sample solution to detection of an electrochemical reaction, and is contained in the sample solution only by mounting the nucleic acid detection cassette 10 containing the sample. Data on the base sequence of nucleic acids can be obtained.

[Basic structure of nucleic acid detection cassette]
As shown in FIG. 2, the nucleic acid detection cassette 10 has a rectangular appearance that is attached to and taken out from the nucleic acid tester 8 in the direction indicated by the arrow 5. Here, the nucleic acid detection cassette 10 is provided with a notch 22 in a part thereof in order to determine the orientation when the nucleic acid detection cassette 10 is attached to the nucleic acid test device 8. In the embodiment shown in FIG. 2, the front left corner of the rectangular nucleic acid detection cassette 10 is cut out with reference to the insertion (mounting) direction 5 into the device, and the cutout portion 22 is provided. The user (operator) can correctly attach the nucleic acid detection cassette 10 to the nucleic acid test device 8 by confirming the notch 22 as the tip side. In addition, the form is not limited to the form in which the corner is cut out in this way, and the upper surface of the rectangular nucleic acid detection cassette 10 may be specified by notching other parts, and the mounting direction of the nucleic acid detection cassette 10 may be specified. In the following description, the side opposite to the tip side is referred to as the rear, and the surface on which the cap 20 is attached to the nucleic acid detection cassette 10 is referred to as the upper surface, with reference to the mounting direction with the notch 22 of the nucleic acid detection cassette 10 as the tip side. ..

As shown in a disassembled manner in FIG. 3, the nucleic acid detection cassette 10 is a laminated structure having a flow path inside. The nucleic acid detection cassette 10 is composed of a lower plate 30, a packing 40, an upper plate 50, a cover plate 60, and a cap 20. The lower plate 30 is made of a hard material, for example, a hard resin material such as polycarbonate, and a chip recess 130 for accommodating the DNA chip 6 is formed, and a flow path is formed so as to include a nucleic acid probe immobilization region. The DNA chip 6 is fitted and fixed in the recess 130 so that the surface of the flow path forming portion 100A, which is a region, faces the upper surface. The lower plate 30 is formed with a groove 132 for a flow path of the liquid delivery system communicating with the detection flow path 100 formed on the DNA chip 6, and a recess 134 for a syringe for defining the liquid feed system. And a tank recess 136 is formed.

The packing 40 is made of an elastic material, for example, a rubber elastic material such as an elastomer, and is placed on the lower plate 30. The packing 40 is formed with a bulging portion 144 for defining a liquid feeding system, a flow path through hole 142, and a vertically long through groove 146 corresponding to the recess 134 of the lower plate 30. Further, the packing 40 is provided with a groove 111 on the DNA chip 6 for defining the detection flow path 100. Further, the packing 40 is provided with opening protrusions 141 and 143 for defining the liquid injection hole, and opening protrusions 145 and 147 for defining the air vent hole are formed. Furthermore, a vertically long through groove 149 is formed to allow the electrode pad portion of the DNA chip 6 to be accessed from the outside.

The upper plate 50 is made of a hard material, for example, a hard resin material such as polycarbonate, is placed on the packing 40, and the packing 40 is pressed and sandwiched between the upper plate 50 and the lower plate 30. More specifically, a plurality of stud pins 156 and 158 are provided on the lower surface of the upper plate 50 so as to project, and the stud pins 156 arranged around the upper plate 50 are peripheral studs provided around the lower plate 30. It is inserted directly into the hole 138A. Further, the stud pin 158 provided on the upper plate 50 so as to face the packing 40 is inserted into the stud hole 138B provided on the upper surface of the lower plate 30 through the insertion hole 148 provided in the central portion of the packing 40. NS. The tips of these stud pins 156 and 158 are thermally caulked, deformed into a retaining boss, and fixed to the lower plate 30. The upper plate 50, packing 40 and lower plate 30 are integrated by the stud pins 156 and 158. In this structure, the upper plate 50 is fixed to the lower plate 30 so as to pressurize the packing 40, a liquid-tight liquid feeding system is defined between the upper plate 50 and the packing 40, and a liquid-tight liquid feeding system is defined between the lower plate 30 and the packing 40. Similarly, a liquid-tight liquid feeding system is defined. The upper plate 50 has a bulge 144 formed in the packing 40, a through hole 142, a vertically long through groove 146, and a vertically long through groove 154, a groove communicating with the through hole 142 (not shown), and a vertically long through groove. A vertically long through groove 159 and the like communicating with the groove 149 are formed. Further, the upper plate 50 is formed with through holes 151, 153, 155, and 157 through which the opening protrusions 141, 143, 145, and 147 of the packing 40 are inserted.

The upper plate 50 is divided into a rear region 150 and a front region 152, and the rear region 150 and the front region 152 are connected via step 169. A cover plate 60 is non-removably mounted in the rear region 150 of the upper plate 50. The cover plate 60 is made of a hard material, for example, a hard resin material such as polycarbonate, and has a notch 160 into which the cap 20 can be fitted. A cap 20 is attached to the notch 160. The cover plate 60 is provided with a vertically elongated groove 164 corresponding to the vertically elongated groove 154 and the like formed in the rear region 150 of the upper plate 50 and having a partition 165.

An engaging protrusion 162 extends downward around the lower surface of the cover plate 60 facing the rear region 150 of the upper plate 50, and the engaging protrusion 162 is provided corresponding to the engaging protrusion 162. Through the insertion hole 166 of the upper plate 50, it is irremovably engaged with the engagement hole 131 provided in the lower plate 30.

(Liquid transfer system in nucleic acid detection cassette)
The liquid feeding system in the nucleic acid detection cassette will be described with reference to FIGS. 4, 5 and 6. FIG. 4 shows through the liquid feeding system provided inside the nucleic acid detection cassette 10. Further, FIGS. 5 and 6 show the arrangement of the liquid feeding system formed in the nucleic acid detection cassette 10 shown in FIG.

As shown in FIG. 4, the nucleic acid detection cassette 10 is composed of a syringe unit 70, an amplification unit 80, and a detection unit 90, and the liquid feeding system is configured to communicate the syringe unit 70, the amplification unit 80, and the detection unit 90. Has been done. The syringe portion 70 is covered with a cover plate 60 and a cap 20 and is provided in the nucleic acid detection cassette 10 corresponding to the rear region 150 of the upper plate 50. The amplification unit 80 and the detection unit 90 are provided in the nucleic acid detection cassette 10 corresponding to the front region 152 of the upper plate 50 and are composed of a flow path.

The syringe section 70 has the same composition as the sample syringe 702 containing the sample solution, the first cleaning solution syringe 704 filled with the first cleaning solution, the inserting agent syringe 706 filled with the inserting agent solution, and the first cleaning solution. A second cleaning solution syringe 708 filled with the second cleaning solution is arranged along the short side direction of the nucleic acid detection cassette 10. These syringes 702, 704, 706 and 708 are formed as a tubular space extending in the longitudinal direction of the nucleic acid detection cassette 10, and each of the tubular spaces has a recess 134 of the lower plate 30 and a bulge 144 of the packing 40. By covering with, it is defined between the recess 134 and the bulge 144.

As shown in FIG. 4, the sample syringe 702 is communicated with the sample injection hole 710 for injecting the sample liquid through the flow path 712 in which the sample liquid flows, and the air bleeding opening 716 is communicated through the air bleeding flow path 714. It is communicated with. Further, the flow path 712 is branched on the inflow side of the sample syringe 702 and communicates with the normally closed valve 718. The first cleaning liquid syringe 704 also communicates with the first cleaning liquid supply hole 720 via the flow path 724, and also communicates with the air vent opening 726 via the flow path branched from the flow path 722 communicating with the normally closed valve 728. Has been done. The first cleaning liquid is pressurized from the first cleaning liquid supply hole 720 and supplied to the first cleaning liquid syringe 704, and the air in the first cleaning liquid syringe 704 is discharged to the outside through the air vent opening 726. Similarly, the insertion agent syringe 706 is also communicated with the insertion agent supply hole 730 via the flow path 734, and also through the flow path branched from the flow path 732 communicating with the normally closed valve 738 to the air vent opening 746. It is communicated. The insertion agent is pressurized from the insertion agent supply hole 730 and supplied to the insertion agent syringe 706, and the air in the insertion agent syringe 706 is discharged to the outside through the air vent opening 736. Further, the second cleaning liquid syringe 708 is also communicated with the second cleaning liquid supply hole 740 via the flow path 724, and the air vent opening 746 is communicated with the flow path 742 communicating with the normally closed valve 748. It is communicated with. The second cleaning liquid is pressurized from the second cleaning liquid supply hole 740 and supplied to the second cleaning liquid syringe 708, and the air in the second cleaning liquid syringe 708 is discharged to the outside through the air vent opening 746. Here, the second cleaning liquid is prepared as a cleaning liquid having the same composition as the first cleaning liquid.

Here, the syringes 702, 704, 706 and 708 have a function as a tank for storing the sample solution, the cleaning solution or the reagent filled in the syringe 702, 704, 706 and 708, and flow the sample solution, the cleaning solution or the reagent filled in the inside thereof. It has the function of a pump that sends out to 712, 722, 732, and 742. That is, the insides of the syringes 702, 704, 706 and 708 are formed in a cavity by the elastic bulging portion 144, and the bulging portion having elasticity is formed by the syringe rod provided in the liquid feed control mechanism 16 of the inspection device 8. By crushing 144, the sample solution, cleaning solution or reagent inside it can be sent out to the flow paths 712, 722, 732, 742. Also, the normally closed valves 718, 728, 738, 748 have the function of maintaining the storage of the sample solution, cleaning solution or reagent in the syringes 702, 704, 706 and 708, and when the test is started, these are normally closed. The closed valves 718, 728, 738, and 748 are sequentially opened by the valve rod provided in the liquid feed control mechanism 16 of the inspection device 8 and are kept open. The supply holes 710, 720, 730, 740 and the air vent openings 716, 726, 736, 746 are formed by the opening protrusions 141, 143, 145, 147 provided on the packing 40, and the sample solution, cleaning solution or reagent is injected into the syringe 702, 704, 706 and 708 are provided for external injection. The supply holes 720, 730, 740 and the air vent openings 726, 736, 746 have a cover plate 60 attached to the top plate after the cleaning solution or reagent has been injected into the syringes 704, 706 and 708. The cover plate 60 compressively deforms and closes the opening protrusions 141 and 147 provided on the packing 40. Therefore, after the cleaning liquid or reagent is injected into the syringes 704, 706 and 708, the cleaning liquid or reagent is prevented from leaking out of the nucleic acid detection cassette 10. Further, the sample injection hole 710 and the air vent opening 716 are closed by compressing and deforming the opening protrusions 143 and 145 of the packing 40 by the cap 20 after the sample solution is injected into the sample syringe 702. Therefore, after the sample solution is injected into the sample syringe 702, the sample solution is prevented from leaking out of the nucleic acid detection cassette 10.

The amplification unit 80 is provided with normally open valves 810 and 820 on the input port side and the output port side. The normally open valves 810 and 820 on the input port side are connected to the starting end side of the amplification flow path 812 of the amplification flow paths 812 and 816 communicating in series with each other, and the normally open valve 820 on the output port side is an amplification flow. It is connected to the terminal side of the road 816. The normally open valve 810 on the input port side is connected to a flow path 802 commonly connected to the normally closed valves 718 and 728 of the syringe portion 70. Further, the normally open valve 820 on the output port side is connected to the flow path 806. The normally closed valves 738 and 748 of the syringe portion 70 are commonly connected to the flow path 804. Further, the flow path 804 and the flow path 806 are commonly connected and communicate with the flow path 908 of the detection unit 90. Here, the output port is a port that goes out from the lower plate to the upper plate, and the input port is a port that goes out from the upper plate and returns to the lower plate.

As shown in an enlarged manner in FIG. 7, the patterns of the amplification channels 812 and 816 are formed in a form in which the meander channels for increasing the channel length are folded back in a U shape. The amplification channels 812 and 816 having the U-shaped meander pattern are arranged line-symmetrically with respect to the central axis 818 between them, and are communicated with each other so as to form a unidirectional flow path. ing. Since the amplification channels 812 and 816 have a symmetrical pattern, the heater of the temperature control mechanism 14 provided in the inspection device 8 can be designed to heat the amplification channels 812 and 816 relatively uniformly. can.

Wells 830 are arranged in the amplification channels 812 and 816 so as to be spaced apart from each other by a certain length or more along the channels. In the example shown in FIG. 4, in the meander-shaped amplification channels 812 and 816, the wells 830 are arranged with a substantially constant flow path length, so that the wells 830 are scattered in a straight line in the amplification section 80. And are arranged. Similarly, the heater of the temperature control mechanism 14 can be designed to heat these wells 830 relatively uniformly because the wells 830 are interspersed in a straight line.

In the plurality of wells 830 provided in the amplification unit 80, different types of primer sets 832 are freely fixed to each well. When a sample solution containing one or more sample DNAs (base sequences) is supplied into the amplification unit 80, the immobilized primer set 832 is released into the sample solution. Then, when the amplification unit 80 is heated by the heater of the temperature control mechanism 14, the plurality of sample DNAs are multi-amplified by the plurality of types of primer sets 832. Here, in each well 830, one kind of primer set composed of a plurality of primers necessary for amplifying one kind of sample DNA of interest is fixed.

When amplifying this sample DNA, the normally open valve 810 on the input port side and the normally open valve 820 on the output port side of the amplification unit 80 are closed by a valve rod provided in the inspection device 8. Therefore, due to the heat given to the amplification unit 80 when the sample DNA is amplified, the expanded sample solution flows out to the flow paths 802 and 806 outside the input port and output port of the amplification unit 80 and is mixed with the reagents and the like. It is possible to prevent such a situation.

A DNA chip 6 enlarged in FIG. 8 is arranged in the detection unit 90. The DNA chip 6 is provided with a detection flow path 100 communicating with a pair of U-shaped flow paths. The detection flow path 100 is provided with a pair of U-shaped grooves 111 that communicate with each other in the packing 40. The groove 111 is superposed on the detection flow path 100 on the DNA chip 6, and the packing 40 is liquid-tightly adhered to the DNA chip 6, so that the detection flow path 100 is defined between the DNA chip 6 and the packing 40. The detection flow path 100 is connected to an input port 910 and an output port 914 on the DNA chip 6. As shown in FIG. 6, the input port 910 is connected to the output port 912 via the flow path 922, and the end of the flow path 908 is connected to the output port 912. Further, the output port 914 is connected to the input port 916 via the flow path 924, and the start end of the flow path 926 is connected to the input port 916. Therefore, the sample solution containing the amplification product produced by the amplification reaction in the amplification unit 80 is detected by the DNA chip 6 from the amplification unit 80 via the flow paths 806 and 908, the output port 912, the flow path 922 and the input port 910. It flows into the road 100.

In the flow path configuration described above, by sending the first cleaning liquid filled in the first cleaning liquid syringe 704 to the amplification channels 812 and 816, the sample solution containing the amplification product is extruded from the amplification channels 812 and 816. It flows into the detection flow path 100 of the DNA chip 6. As shown in FIG. 6, after that, the inserting agent and the second cleaning liquid filled in the syringes 706 and 708 are supplied to the flow path 804 via the flow path 732 and the flow path 742, respectively. The inserting agent or the second cleaning liquid supplied to the flow path 804 flows into the detection flow path 100 of the DNA chip 6 via the flow path 908, the output port 912, the flow path 922, and the input port 910.

In particular, the sample solution containing the amplification product is pushed out to the flow path 908 where the flow paths 804 are merged via the amplification flow paths 812 and 816 of the amplification unit 80 by sending the first washing liquid, and the detection flow path of the DNA chip 6. It has flowed into 100. As for the delivery of the first cleaning liquid, the sample solution can be reliably delivered from the amplification channels 812 and 816 of the amplification unit 80 to the detection flow path 100 of the DNA chip 6. Moreover, the flow path 804 into which the inserting agent and the second cleaning agent flow in is separated from the amplification flow paths 812 and 816 and merges with the flow path 908. Therefore, the insertion agent and the second cleaning agent are prevented from flowing into the detection flow path 100 without passing through the amplification passages 812 and 816, and the insertion agent is prevented from being mixed with the sample solution of the amplification unit 80, and at the same time, the residue in the amplification unit 80 remains. It is also prevented from being heated by heat.

The detection unit 90 is provided with a waste liquid tank 930 and an auxiliary waste liquid tank 932 for storing the waste liquid from the DNA chip 6. The waste liquid tank 930 communicates with the DNA chip 6 via the output port 914, the flow path 924, the input port 916, and the flow path 926. Further, the waste liquid tank 930 communicates with the auxiliary waste liquid tank 932 via the flow path 926. The auxiliary waste liquid tank 932 is preferably provided with an exhaust hole 934 for exhausting the air in the auxiliary waste liquid tank 932, and when the waste liquid flows into the waste liquid tank 930 from the DNA chip 6, the air in the auxiliary waste liquid tank 932 is provided. Is exhausted.

When the second cleaning liquid is sent from the second cleaning liquid syringe 708, the unreacted sample in the detection flow path 100 is pushed out as waste liquid from the detection flow path 100 by the second cleaning liquid and flows into the waste liquid tank 930. After that, when the insertion agent is sent from the insertion agent syringe 706, the second cleaning liquid in the detection flow path 100 is extruded as waste liquid from the detection flow path 100 by this insertion agent, and similarly flows into the waste liquid tank 930. NS.

Here, the waste liquid generated in the series of inspection steps flows into the waste liquid tank 930 and is retained in the waste liquid tank 930, and hardly flows into the auxiliary waste liquid tank 932, or a small amount of waste liquid flows through the flow path 926. Inflow through. Moreover, the supply holes 710, 720, 730, 740 and the air vent openings 716, 726, 736, 746 are closed after the sample solution is injected into the sample syringe 702, and these supply holes 710, 720, 730, 740 and The liquid feeding system between the air vent openings 716, 726, 736, 746 and the exhaust hole 934 of the auxiliary waste liquid tank 932 is maintained liquid tightly. Therefore, even if the liquid feeding system is heated in a series of inspection steps, it is possible to prevent the waste liquid from leaking from the exhaust hole 934 of the auxiliary waste liquid tank 932.

These waste liquid tanks 930 and 932 have a vertically long recess (not shown) defined by a recess 136 formed in the lower plate 30, a vertically long through groove 146 of the packing 40, and a recess 136 formed on the back surface of the upper plate 50. It is formed as a space. In particular, the waste liquid tank 930 preferably has a volume capable of receiving all of the sample solution, the first cleaning liquid, the inserting agent and the second cleaning liquid extruded from the syringes 702, 704, 706 and 708.

The flow paths 712, 714, 722, 724, 732, 734, 742, 744, 802, 804, 806, 908, 926 in the liquid feeding system described above cover the groove 132 formed in the lower plate 30 with the packing 40. It is a flow path defined between the groove 132 and the flat lower surface of the packing 40. Similarly, the amplification channels 812 and 816 are also defined between the groove 132 and the flat lower surface of the packing 40 by covering the meander-shaped groove 132 having the wells 830 formed in the lower plate 30 with the packing 40. It is a flow path. The detection flow path 100 of the DNA chip 6 has a U-shaped flow path defined by an electrode arrangement on a flat substrate made of glass or silicon. The U-shaped flow path on the substrate is aligned with the U-shaped groove 111 provided in the packing 40, and by covering the flat substrate with the packing 40, the detection flow path 100 becomes the U-shaped groove of the packing 40. It is a flow path defined between the flat upper surface of the substrate and the flat upper surface of the substrate.

Ports 910 and 914 of the DNA chip 6 correspond to through holes (corresponding to reference numerals 910 and 914) drilled in the packing 40. Further, the output port 912 of the flow path 908 and the input port 916 of the flow path 926 also correspond to through holes (corresponding to reference numerals 912 and 916) formed in the packing 40. The flow path 922 between the output port 912 and the port 910 and the flow path 924 between the input port 916 and the port 914 are formed by covering the groove (not shown) formed on the lower surface of the upper plate 50 with the packing 40. It is defined between this groove and the flat upper surface of the packing 40. Therefore, the flow path leading to the ports 910 and 914 of the DNA chip 6 communicates with each other on the upper surface side of the DNA chip 6. At the ports 910 and 914 of the DNA chip 6, the upper plate 50 is pressed toward the lower plate 30, and the packing 40 is provided between the two so as to be liquid-tight. Therefore, even if the DNA chip 6 has a hard substrate structure, the DNA chip 6 is maintained in close contact with the packing 40, and the detection flow path 100 of the DNA chip 6 is between the lower plate 30 and the packing 40. It is surely communicated with the formed liquid transfer system flow path. Even if the detection flow path 100 of the DNA chip 6 is heated, it is possible to prevent the sample solution or the like from leaking from the detection flow path 100 of the DNA chip 6.

[DNA chip]
The DNA chip 6 will be described with reference to FIGS. 3 and 8. As shown in FIG. 3, the DNA chip 6 has a detection flow path 100 defined so as to cover a flow path forming portion 100A on a flat surface of a glass or silicon substrate by a groove 111 on the back surface of the packing 40, and the detection thereof. It has electrode pad regions 110 and 112 arranged on both sides of the flow path 100. As shown in FIG. 9, the detection flow path 100 is provided with working electrodes 940 at regular intervals. Further, electrode pads 942 are arranged in the electrode pad regions 110 and 112, and the electrode pads 942 are electrically connected to the corresponding working electrodes 940, respectively. Further, a reference electrode 944 and counter electrodes 946 and 948 are provided on the U-shaped top of the detection flow path 100, and are connected to the corresponding electrode pads 942, respectively. In FIG. 9, for the purpose of simplifying the drawing, the connection between the electrode pad 942 and the working electrode 940, the reference electrode 944, and the counter electrodes 946 and 948 is omitted.

The action electrode 940 is a nucleic acid for detecting a nucleic acid probe containing a sequence complementary to the base sequence of the nucleic acid to be detected, for example, SNP (1) to SNP (N) (single nucleotide polymorphism: Single Nucleotide Polymorphism). The probe is fixed to an electrode, for example, a gold electrode for each type. A plurality of nucleic acid probes are fixed for each type of working electrode 940. Under heating, if there is a complementary base sequence (ie, target DNA) in the sample solution, it will be bound by a hybridization reaction to the immobilized nucleic acid probe. The unreacted sample solution that has not been bound by the hybridization reaction is pushed out from the detection flow path 100 by the second cleaning solution and removed, and the working electrode 940 is washed with the second cleaning solution. After that, the inserting agent is supplied to the detection flow path 100 to remove the second cleaning liquid. With the working electrode 940 immersed in the insert, a constant voltage is applied between the working electrode 940 and the counter electrodes 946 and 948. Here, the insertion agent contains a substance that recognizes and enters the double-stranded portion generated by the hybridization reaction on the working electrode 940 and is electrochemically activated. A signal current is detected from the working electrode 940 to which the nucleic acid probe which has become double-stranded by the hybridization reaction due to the application of voltage is fixed. This signal current is detected when the current probe included in the measuring unit 12 shown in FIG. 1 described above is physically contacted with the electrode pad 942. The detected current is amplified and stored in the buffer of the measuring unit 12, and is output as detection data by the external computer 4.

Here, the reference electrode 944 has an action of monitoring the applied voltage between the working electrode 940 and the counter electrodes 946 and 948 and making the applied voltage between the working electrode 940 and the counter electrodes 946 and 948 constant via the feedback circuit. doing.

(Top structure of nucleic acid detection cassette)
The upper surface structure of the nucleic acid detection cassette 10 will be described in more detail with reference to FIG. 2 again and with reference to FIG. 9 newly. FIG. 9 shows the appearance of the cover plate 60 removed from the nucleic acid detection cassette 10 shown in FIG.

As described with reference to FIGS. 2 and 3, a cover plate 60 fitted and fixed to the nucleic acid detection cassette 10 is provided in the rear region of the nucleic acid detection cassette 10, and the cover plate 60 has a cap 20. It is installed. The cap 20 is formed in an L shape having a rectangular upper surface flat portion 21 and a side portion 23 extending from the upper surface flat portion 21 along the rear side surface of the nucleic acid detection cassette 10. Further, a shaft portion 25 for opening and closing the cap 20 is provided on the side portion of the flat portion 21, and the side portion 23 is engaged and fixed to the engaging groove (not shown) of the lower plate 30. 27 is provided. The cover plate 60 is formed with a notch 160 for receiving the flat portion 21 of the cap 20, and a shaft support hole (not shown) for axially supporting the shaft portion 25 of the cap 20 is a notch in the flat portion 21. It is formed on the side surface of the part. When the cap 20 is closed with the shaft portion 25 as a fulcrum, the cap 20 connects the rectangular upper surface flat portion 21 of the cap 20 to the upper surface of the cover plate 60 to give a substantially flat surface to the nucleic acid detection cassette 10. It is fitted in 10. When the cap 20 is pressed against the cover plate 60 at the time of this fitting, the engaging portion 27 of the cap 20 is engaged with the engaging groove (not shown) of the lower plate 30. As a result, the cap 20 is irremovably fixed to the lower plate 30. After the sample solution is injected into the sample syringe 702 through the sample injection hole 710, when the cap 20 is fitted and attached, the sample injection hole 710 and the air vent opening 716 are pressure-welded and deformed on the lower surface of the cap 20 to form the sample injection hole. The 710 and the air vent opening 716 are closed. Therefore, the nucleic acid detection cassette 10 is hermetically sealed to prevent the sample from leaking to the outside from the nucleic acid detection cassette 10 even if the nucleic acid detection cassette 10 is heated in the amplification of the sample or the detection of the electrochemical reaction of the sample. Will be done.

In this way, before filling the nucleic acid detection cassette 10 with the sample, the cap 20 is opened with respect to the cover plate 60 while being pivotally supported by the cover plate 60 at the shaft portion 25, and is around the shaft portion 25. It is installed in a state where it is maintained rotatably. After the nucleic acid detection cassette 10 is filled with the sample, the cap 20 is irremovably fixed to the nucleic acid detection cassette 10 as described above. The non-removable fixation of the cap 20 to the nucleic acid detection cassette 10 makes it clear to the user that the sample is stored in the nucleic acid detection cassette 10, and the sample is erroneously injected into the nucleic acid detection cassette 10 again. It is possible to prevent such a situation.

Vertical grooves 164 are arranged in parallel on the cover plate 60 along the width direction of the nucleic acid detection cassette 10, and syringes 702, 704, 706, and 708 bulge into the vertical grooves 164 inside the cover plate 60. It is provided. Further, the cover plate 60 is provided with T-shaped recesses 41, 42, 44, 46 along the width direction of the nucleic acid detection cassette 10, and the normally closed valves 718, 728 are provided behind the recesses 41, 42, 44, 46. , 738, 748 cantilever beam structures are provided. When the cantilever beam structure is opened from the back side of the cover plate 60 by the valve rod of the liquid feed control mechanism 16, the normally closed valves 718, 728, 738, 748 behind the recesses 41, 42, 44, 46 are opened. Be released. The vertical groove 164 in which the syringes 702, 704, 706, and 708 are arranged has a width so that a finger cannot enter, and the vertical groove 164 having a large vertical length is provided with a partition 165 as shown in FIG. It is designed to prevent the entry of fingers in the longitudinal direction.

The nucleic acid detection cassette 10 shown in FIG. 2 is provided with uneven grooves 139 for slip prevention on both side surfaces in the width direction of the upper plate 50 and the lower plate 30 provided with the cover plate 60, so that the user can reliably detect nucleic acids. It is possible to grip the cassette 10.

The cover plate 60 of the nucleic acid detection cassette 10 shown in FIG. 2 is attached and fixed to the upper plate 50 so that the upper surface of the cover plate 60 is flush with the front region 152. A rectangular recess 51 is formed in the front region 152 of the upper plate 50 to fix the amplification portion region, and rod holes 52 and 54 for operating the normally open valves 810 and 820 are provided on both sides of the recess 50. ing. The valve rod of the liquid feed control mechanism 16 is inserted into the rod holes 52 and 54, and the tip of the valve rod is pressed against the normally open valves 810 and 820 to open the normally open valves 810 and 820. Further, vertically elongated probe holes 56 and 58 for mounting the current probe are formed in parallel with the front region 152 of the upper plate 50 in the electrode pad regions 110 and 112 in which the electrode pads of the DNA chip 6 are arranged.

In the rear region 150 of the upper plate 50 where the cover plate 60 is removed and exposed, openings for the first cleaning liquid supply hole 720, the insertion agent supply hole 730, and the second cleaning liquid supply hole 740 provided in the packing 40 are provided. The tip of the protrusion 141 and the tip of the opening protrusion 147 for the air vent openings 726, 736, 746 are exposed. In the manufacturing process of the nucleic acid detection cassette 10, the first cleaning liquid is injected into the first cleaning liquid syringe 704 through the first cleaning liquid supply hole 720 with the cover plate 60 as shown in FIG. 9 removed, and the insertion agent is supplied. The insertion agent solution is injected into the insertion agent syringe 706 through the hole 730, and the second cleaning solution is injected into the second cleaning solution syringe 708 through the second cleaning solution supply hole 740. After that, the cover plate 60 is irremovably attached and fixed to the rear region 150 of the upper plate 50. At the time of this mounting and fixing, the lower surface of the cover plate 60 is pressed against the opening protrusion 141 and the opening protrusion 147, and the opening protrusion 141 and the opening protrusion 147 are pressed against the lower surface of the cover plate 60. Therefore, when the cover plate 60 is attached to the upper plate 50, the first cleaning liquid supply hole 720, the insertion agent supply hole 730, the second cleaning liquid supply hole 740, and the air vent openings 726, 736, and 746 are closed.

In the manufacturing process of the nucleic acid detection cassette 10, the bulging portions of the syringes 702, 704, 706, and 708 are used for the sample solution, the first cleaning solution, the insertion agent solution, and the second cleaning solution for the syringes 702, 704, 706, and 708. 144 is crushed. After that, the negative pressure generated in the syringes 702, 704, 706, and 708 when the bulging portion 144 returns is utilized, and the sample solution, the first cleaning liquid, the insertion agent liquid, and the second cleaning liquid are used in the syringes 702, 704, and 706. It is injected into the 708.

(Structure of normally open valve)
As described above, the amplification unit 80 is provided with normally open valves 810 and 820 on the input port side and the output port side of the amplification flow paths 812 and 816. The normally open valves 810 and 820 are formed as a tubular portion 55 of the flexible packing 40 in the rod holes 52 and 54 of the upper plate 50 as shown in FIGS. 10 and 11. The packing 40 is formed with a recess 57 corresponding to the rod holes 52 and 54 of the upper plate 50, and the tubular portion 55 is arranged in the recess 57. A cavity (flow path) for the normally open valves 810 and 820 is formed between the tubular portion 55 and the upper surface of the lower plate 30 on which the tubular portion 55 is arranged, and this cavity is formed in the flow path 802. And the amplification flow path 812 or the amplification flow path 816 and the flow path 806 are communicated with each other. As is clear from this structure, since the tubular portion 55 is integrally formed with the packing 40 in the recess 57, it is sufficiently thinner than the film thickness T1 of the packing 40, and more preferably the film thickness T2 (T1). > T2) is formed thinner. Here, the film thickness T1 corresponds to the convex region film thickness of the packing 40 formed in the convex region and the concave region region, and the film thickness T2 corresponds to the concave region film thickness. Therefore, as shown in FIG. 11, the hollow portion provided inside the tubular portion 55 can be crushed by the rod 59 inserted from the outside, and the hollow portion can be closed by crushing the tubular portion 55. can. By closing the cavity, the flow path between the flow path 802 and the amplification flow path 812 or the flow path between the amplification flow path 816 and the flow path 806 is blocked. Further, since the tubular portion 55 has flexibility, the crushed tubular portion 55 is removed by retracting the rod 59 after the amplification portion 80 is heated to amplify the target DNA in the sample. It can be returned to the original cylindrical part in which the cavity is formed. Therefore, the flow path 802 and the amplification flow path 812 or the amplification flow path 816 and the flow path 806 can communicate with each other through the cavity in the tubular portion 55.

(Normally closed valve structure)
The normally closed valves 718, 728, 738, and 748 are configured such that the normally opened valve 57 shown in FIG. 11 is closed by a protrusion 43 provided on the back surface of the cover plate 60. More specifically, the normally closed valves 718, 728, 738, and 748 are entered into the groove provided in the packing 40 through the through hole provided in the upper plate 50 by the protrusion 43, and the flexible cylinder in the groove is entered. It is configured to crush the shaped portion and block the flow path of the tubular portion. The protrusion 43 provided on the back surface of the cover plate 60 is formed in a cantilever structure in which the base is pivotally fixed to the cover plate 60 and the tongue piece extending from the base is movably supported by the cover plate 60. When the normally closed valves 718, 728, 738, and 748 are opened, the tongue piece of the cantilever structure is lifted by the rod, the protrusion 43 is removed from the tubular part, and a flow path is secured in the tubular part. Be released.

(Packing structure)
The structure of the packing 40 will be described with reference to FIG. FIG. 12 shows the back shape of the packing 40. On the back surface of the packing 40, the bulging portion 144 is shown as a recess, and through holes 141A, 143A, 145A, and 147A are formed at locations corresponding to the opening protrusions 141, 143, 145, and 147. Further, the packing 40 is provided with an insertion hole 148 into which a stud pin 158 provided on the lower surface of the upper plate 50 is inserted. The back surface of the packing 40 is formed in a shape having an uneven region corresponding to the flow path system. The convex region on the back surface of the packing 40 has a relatively thick film thickness T1, and the concave region between the convex regions has a film thickness T2 thinner than the film thickness T1. Specifically, a frame portion 144B having a thick film thickness T1 is formed on the back surface of the packing 40 so as to surround the bulge portion 144 that defines the syringes 702, 704, 706, and 708. The circumferences of the through holes 141A, 143A, 145A, and 147A are also formed in ring-shaped protrusions 141B, 143B, 145B, and 147B having a film thickness of T1. Further, a frame portion 146B having a thick film thickness T1 is formed around the vertically long through groove 146 that defines the tanks 930 and 932 together with the recess 136 of the lower plate 30. Furthermore, the strip-shaped regions 712B, 714B to which the grooves 132 of the lower plate 30 are closely attached, which define the flow paths 712, 714, 722, 724, 732, 734, 742, 744, 802, 804, 806, 908, 926, 722B, 724B, 732B, 734B, 742B, 744B, 722B, 732B, 742B, 802B, 804B, 806B, 908B are also formed to have a thick film thickness T1. Therefore, the lower plate 30 is closely adhered to the region having the film thickness T1 in a liquid-tight manner, and the liquid-tight sealing of the liquid feeding system can be ensured.

The rectangular regions 812B and 814B of the packing 40 corresponding to the amplification channels 812 and 816 of the amplification unit 80 are also formed in a rectangular flat region that can cover the amplification passages 812 and 816 of the amplification unit 80, and this flat region is also formed. It has a thicker film thickness T1 than the film thickness T2 in the peripheral region. Therefore, the amplification channels 812 and 816 of the amplification section 80 formed on the lower plate 30 are also closely adhered to the region where the lower plate 30 has the film thickness T1 to ensure the liquidtight sealing of the amplification passages 812 and 816. Can be secured. This liquid-tight sealing can sufficiently withstand the heating of the amplification unit 80.

Furthermore, a pair of U-shaped through grooves 102 connected to each other are formed in the flow path forming region 100B of the packing 40 corresponding to the detection flow path 100 of the DNA chip 6. The packing 40 is brought into close contact with the flat substrate of the DNA chip 6 so that the through groove 102 of the flow path forming region 100B of the packing 40 forms the detection flow path 100 of the DNA chip 6. Here, the working electrode 940, the reference electrode 944, and the counter electrodes 946 and 948 are brought into close contact with each other by aligning the packing 40 with respect to the DNA chip 6 so as to be arranged in the groove 102. Similarly, the flow path forming region 100B of the packing 40 has a film thickness T1 that is thicker than the film thickness T2 in the surrounding region. Therefore, when the lower plate 30 is brought into close contact with the flow path forming region 100B of the packing 40 having the film thickness T1, the detection flow path 100 can be formed liquid-tightly on the DNA chip 6.

Further, when the upper plate 50 is crimped to the lower plate 30 via the packing 40, the flexible packing 40 is deformed, but the thickness change due to the deformation changes from the convex region to the concave region. It is absorbed by the protrusion.

The frame portion 144B defining the syringes 702, 704, 706 and 708 has a thick film thickness T1 as described above, but more preferably, a slit 144C penetrating the packing 40 is formed in the region between the frame portions 144B. Is formed. The slit 144C reliably allows the flexible packing 40 to be deformed when the upper plate 50 is crimped to the lower plate 30 via the packing 40, removes the strain generated in the packing 40, and removes the strain generated in the packing 40. And the packing 40 can be more reliably adhered to each other. Further, the region having the film thickness T2 (thin film region) provided on the outer periphery of the syringes 702, 704, 706 and 708 to absorb the change in thickness due to the deformation of the packing 40 is cut and removed as necessary. Is also good.

Similarly, it is preferable that slits or through holes 812C and 816C are provided between the regions 812B and 814B of the packing 40 corresponding to the amplification channels 812 and 816 and on both sides thereof. The slits or through holes 812C and 816C can remove the strain generated in the packing 40 at the time of close contact.

The insertion hole 148 into which the stud pin 158 is inserted is preferably formed to have a diameter larger than the diameter of the stud pin 158 in order to absorb the deformation of the packing 40, and the periphery thereof is formed into a cylindrical shape having a film thickness T2. Has been done. When the upper plate 50 is crimped to the lower plate 30 by heat caulking the stud pin 158, the tubular portion of the insertion hole 148 is deformed and enters the insertion hole 148 to securely hold the stud pin 158 and the upper plate. The 50 can be securely fixed to the lower plate 30.

(Assembly of nucleic acid detection cassette)
The process of assembling the nucleic acid detection cassette shown in FIG. 2 will be described with reference to FIGS. 13, 14 and 15.

First, as shown in FIG. 13, the lower surface of the upper plate 50 is arranged so as to face upward, and a packing 40 to be attached to the lower surface of the upper plate 50 is prepared. A stud pin 158 provided on the lower surface of the upper plate 50 is inserted into an insertion hole 148 provided in the packing 40, and the packing 40 is placed on the lower surface of the upper plate 50 as shown in FIG. Here, the stud pins 156 provided on the outer periphery of the upper plate 50 are arranged around the packing 40 as shown in FIG.

Next, the assembly of the upper plate 50 and the packing 40 shown in FIG. 14 is turned upside down, and as shown in FIG. 15, the packing 40 and the upper plate 50 are placed on the upper surface of the lower plate 30 on which the DNA chip 6 is placed. It will be placed. The stud pins 156 and 158 of the assembly are inserted into the stud holes 138 of the lower plate 30. After that, the upper plate 50, the packing 40, and the lower plate 30 are integrated by heat caulking the tips of the stud pins 156 and 158 to the lower plate 30.

(Inspection device)
FIG. 16 shows the internal structure of the inspection device to which the nucleic acid detection cassette 10 shown in FIG. 2 is mounted. In this inspection device, on the back side of the mounted nucleic acid detection cassette 10, a heater 850 for heating the amplification unit 80 and a Perche element 852 for maintaining the detection flow path 100 of the DNA chip 6 at the reaction temperature are provided. The lift mechanisms 854 and 856 are provided so as to be movable up and down as indicated by arrows. The heater 850, the Perche element 852, and the lift mechanism 854, 856 constitute the temperature control mechanism 14 shown in FIG.

Further, on the upper surface side of the nucleic acid detection cassette 10, a current probe 960 that is electrically connected to the electrode pads 110 and 112 of the DNA chip 6 to take out the detection current is provided so as to be movable up and down. The current probe 960 is electrically connected to a circuit board 962 including a processor for signal processing, and constitutes a measuring unit 12 shown in FIG.

Further, on the upper surface side of the nucleic acid detection cassette 10, a syringe rod 750 for sending out a sample solution, a cleaning solution or an insertion agent solution from the syringes 702, 704, 706 and 708 is provided, and the normally open valves 810 and 820 are closed. A normally open valve rod 59 for this purpose is provided. On the lower surface side of the nucleic acid detection cassette 10, a normally closed valve rod 752 for opening the normally closed valves 718, 728, 738 and 748 is provided. The syringe rod 750, the normally open valve rod 59, and the normally closed valve rod 752 constitute a liquid feed control mechanism 16.

Each of the measuring units 12, the temperature control mechanism 14, and the liquid feeding control mechanism 16 is controlled by a program stored in the control unit 18 to perform an electrochemical inspection of the sample shown in FIG.

(Electrochemical inspection of sample)
The electrochemical inspection of the sample in the nucleic acid testing apparatus shown in FIG. 1 will be described with reference to the flowchart shown in FIG. 17 and the liquid feeding operation shown in FIGS. 18 to 21.

At the time of manufacturing the nucleic acid detection cassette 10, the first cleaning solution syringe 704 is filled with the first cleaning solution, the insertion agent syringe 706 is filled with the insertion agent solution, and the second cleaning solution syringe 708 is filled with the second cleaning solution. Is prepared. Then, the user (operator) injects the sample solution into the sample syringe 702. After that, the nucleic acid detection cassette 10 is attached to the nucleic acid test device 8 and the test is started (step S10). When injecting the sample solution into the sample syringe 702, the user (operator) grasps the nucleic acid detection cassette 10 and injects the sample solution into the sample injection hole 710 using a microsyringe, a medical syringe, or the like. When the injection of the sample solution into the sample syringe 702 is completed, the cap 20 is attached to the nucleic acid detection cassette 10. By attaching the cap 20 to the nucleic acid detection cassette 10 in a non-removable manner, the sample injection hole 710 and the air vent opening 716 are closed.

When the nucleic acid detection cassette 10 is attached to the inspection device 8, the current probe 960 is lowered toward the nucleic acid detection cassette 10 and is electrically contacted with the electrode pads 942 in the electrode pad regions 110 and 112 of the DNA chip 6. The reaction current in the detection flow path 100 can be detected.

When the inspection is started, first, the normally closed valve rod 752 is pressed against the normally closed valve 718 that communicates with the sample syringe 702, and the normally closed valve 718 is opened. After that, the sample syringe 702 is crushed by the syringe rod 750, and the sample solution inside the sample syringe 702 is supplied to the amplification channels 812 and 816 via the flow path 802 as shown in FIG. 18 (step S12). When the amplification channels 812 and 816 are filled with the sample solution, the normally open valve rod 59 crushes the normally open valves 810 and 820 to close the lot holes 52 and 54. Further, the heater 850 is pressed against the amplification unit 80 of the nucleic acid detection cassette 10, and the amplification channels 812 and 816 of the amplification unit 80 are heated by the heater and maintained at a constant temperature. Therefore, in the closed amplification channels 812 and 816, the plurality of types of target DNA contained in the sample solution are multi-amplified by the corresponding plurality of types of primer sets released from the wall surfaces of the amplification channels 812 and 816 ( Step S14).

Next, the normally open valve rod 752 separates from the normally open valves 810 and 820 and returns to the original position, so that the normally open valves 810 and 820 are opened again. Subsequently, the normally closed valve rod 752 is pressed against the normally closed valve 728 that communicates with the first cleaning liquid syringe 704, and the normally closed valve 728 is opened. After that, the first cleaning liquid syringe 704 is crushed by the syringe rod 750, and the first cleaning liquid inside the first cleaning liquid syringe 704 is supplied to the amplification flow paths 812 and 816 via the flow path 802 as shown in FIG. At this time, the sample solution containing the amplification product in the amplification channels 812 and 816 is supplied to the detection channel 100 of the detection unit 90 via the channels 806, 908 and 922 (step S16). Here, the first cleaning liquid pushes out the air in the flow path 722, and the sample solution containing the amplification product is sent from the amplification flow paths 812 and 816 to the detection flow path 100. Therefore, the sample solution containing the amplification product and the first cleaning liquid come into contact with each other via the air layer, and are sent in the flow paths 802, the amplification flow paths 812, and 816 without being mixed with each other.

When the sample solution containing the amplification product is sent to the detection flow path 100, the Perche element 852 of the nucleic acid test device 8 is raised toward the nucleic acid detection cassette 10 and comes into contact with the detection unit 90. Then, the detection flow path 100 is controlled by the Perche element 852 to the optimum temperature for the hybridization reaction. In the detection flow path 100, the target DNA contained in the amplification product in the sample solution binds to the nucleic acid probe by hybridization (step S18). After that, the normally closed valve rod 752 is pressed against the normally closed valve 748 communicating with the second cleaning liquid syringe 708, and the normally closed valve 748 is opened. After that, the second cleaning liquid syringe 708 is crushed by the syringe rod 750, and the second cleaning liquid inside thereof is supplied to the detection flow path 100 via the flow paths 804, 908, and 922 (as shown in FIG. 20). Step S20). Therefore, the sample solution containing the unhybridized unreacted DNA in the detection flow path 100 is sent from the detection flow path 100 to the waste liquid tank 930 via the flow paths 924 and 926. Here, the detection flow path 100 is controlled to the optimum temperature for cleaning by the Perche element 852, and the inside of the detection flow path 100 is cleaned (step S22). When the second cleaning liquid is supplied to the flow path 804, the second cleaning liquid pushes out the sample solution through the air layer. Here, the flow path 804 and the flow path 806 are communicated with each other by the flow path 908, but the liquid feed pressure is applied to the flow path 804 and the flow path 908 via the air layer, and the flow path 806 is back-fed. Pressure is applied through the air layer. Therefore, the first cleaning liquid is not mixed in the second cleaning liquid, the second cleaning liquid is supplied to the detection flow path 100, and the sample solution is sent from the detection flow path 100 to the waste liquid tank 930.

When cleaning is complete, the normally closed valve rod 752 is pressed against the normally closed valve 738 that communicates with the insert syringe 706, and the normally closed valve 738 is opened. After that, the insertion agent syringe 706 is crushed by the syringe rod 750, and the insertion agent liquid inside the insertion agent syringe 706 is supplied to the detection flow path 100 via the flow paths 804, 908, and 922 as shown in FIG. 21 (step). S24). Here, the liquid feed pressure is applied to the flow path 804 and the flow path 908 via the air layer, and the reverse feed pressure is applied to the flow path 806 via the air layer. Therefore, the first cleaning liquid is not mixed with the insertion agent liquid, and the insertion agent liquid is supplied to the detection flow path 100. Further, the second cleaning liquid in the detection flow path 100 is sent from the detection flow path 100 to the waste liquid tank 930 via the flow paths 924 and 926.

After the insertion agent liquid is supplied to the detection flow path 100, the detection flow path 100 is controlled to the optimum temperature for the insertion agent reaction by the Perche element 852, and the insertion agent reaction occurs in the detection flow path 100 (step S26). .. Here, in the insertion agent reaction, for example, the double-stranded portion is recognized by the insertion agent, the insertion agent enters the recognized double-stranded portion, is electrochemically activated, and an electric signal is generated. This insertion agent reaction is detected by the current probe 960 via the electrode pads 942 in the electrode pad regions 110 and 112 of the DNA chip 6 under the application of a voltage (step S28). The measured current detected by the current probe 960 is processed by the processor of the circuit board 962, sent to the computer 4 as measurement data, and analyzed.

As described above, according to the embodiment, it is possible to provide a nucleic acid detection cassette that is highly integrated and can be used for a nucleic acid test apparatus that is automated from sample amplification to detection of sample electrochemical reaction. ..

4 ... Syringe, 6 ... DNA chip, 8 ... Nucleic acid testing device, 10 ... Nucleic acid detection cassette, 12 ... Measuring unit, 14 ... Temperature control mechanism, 16 ... Liquid feeding control mechanism, 18 ... Device control unit, 20 ... Cap, 21 … Rectangular top surface flat part, 23… side part, 22… notch part, 25… shaft part, 27… engaging part, 30… lower plate, 40… packing, 50… upper plate, 51… recess, 52, 54… rod Hole, 55 ... Cylindrical part, 57 ... Indentation, 56, 58 ... Probe hole, 59 ... Rod, 60 ... Cover plate, 70 ... Syringe part, 80 ... Amplification part, 90 ... Detection part, 100 ... Detection flow path, 100B ... Flow path forming region, 110, 112 ... Electrode pad region, 130 ... Chip recess, 131 ... Engagement hole, 132 ... Groove, 134 ... Syringe recess, 136 ... Tank recess, 138 ... Stud hole, 139 ... Concavo-convex Grooves, 144 ... bulges, 144C ... slits, 141, 143, 145, 147 ... opening protrusions, 141A, 143A, 145A, 147A ... through protrusions, 142 ... through holes for liquid feed flow paths, 144B, 146B ... frame parts , 146, 149 ... Vertical through groove, 148 ... Insertion hole, 150 ... Front area, 151, 153, 155, 157 ... Through hole, 152 ... Front area, 155 ... Stud pin, 154, 159 ... Vertical through groove, 156, 158 ... Stud pin, 169 ... Step, 160 ... Notch, 162 ... Engagement protrusion, 164 ... Vertical groove, 166 ... Insertion hole, 702 ... Sample syringe, 704 ... First cleaning liquid syringe, 706 ... Insert agent syringe, 708 ... Second cleaning solution syringe, 710 ... sample injection hole, 720 ... first cleaning solution supply hole, 730 ... insertion agent supply hole, 740 ... second cleaning solution supply hole, 712, 714, 722, 724, 732, 734, 742, 744 ... Channels, 718, 728, 738, 748 ... normally closed valves, 750 ... syringe rods, 752 ... normally closed valve rods, 716, 726, 736, 746 ... air vent openings, 802, 804,806 ... flow paths, 810, 820. ... normally open valve, 812, 816 ... amplification flow path, 830 ... well, 832 ... primer set, 902 ... flow path, 910, 912 ... input port, 914, 916 ... output port, 908, 922, 924, 926, 928. ... Flow path, 712B, 714B, 722B, 724B, 732B, 734B, 742B, 744B, 722B, 732B, 742B, 802B, 804B, 806B, 908B ... Strip region, 812B, 814B ... Rectangular region, 812C, 816 C ... Through hole, 850 ... Heater, 852 ... Perche element, 854, 856 ... Lift mechanism, 930 ... Waste liquid tank, 932 ... Auxiliary waste liquid tank, 940 ... Working electrode, 944 ... Reference electrode, 946, 948 ... Opposite electrode, 960 … Current probe, 962… Circuit board

Claims (8)

The first, second, which are formed to be deformably formed from the outside by a first plate made of a hard material and a packing made of an elastic material, and store a sample solution, a first cleaning solution, an insertion agent solution, and a second cleaning solution, respectively. With the 3rd and 4th syringes,
An injection unit capable of injecting a sample solution into the first syringe from the outside,
An amplification flow path having an input side and an output side is provided, and the amplification flow path is an amplification portion formed by the first plate and the packing, and the amplification flow path has a plurality of wells. Displaced at intervals, different types of primer sets are immobilized in the plurality of wells, and the nucleic acids in the sample solution are released by releasing the primer set into the sample solution supplied into the amplification flow path. And the amplification part where the amplification product is generated by amplification
First and second valves provided on the input side and the output side of the amplification flow path, respectively, and the first and second valves that confine the sample solution in the amplification section during the nucleic acid amplification in the amplification section. 2 valves and
It has a plurality of electrodes and a plurality of electrode pads connected to the plurality of electrodes, which are placed on the first plate, and the plurality of electrodes electrically perform a hybridization reaction corresponding to the amplification product. A chip containing a working electrode to which a plurality of types of nucleic acid probes are fixed for detection is formed between the chip and the packing, and the plurality of electrodes are arranged and have an input side and an output side. A detection channel is provided, and the sample solution containing the amplification product is supplied from the amplification unit, and is formed between the detection unit for detecting the amplification product and between the first plate and the packing. The first, second, third, fourth and fifth channels,
The first and second channels are communicated with the first and second syringes, respectively, so that the sample solution is supplied and then the first cleaning solution is supplied to the amplification section. It is connected in common with the first valve on the input side of the amplification flow path.
The fifth flow path is connected to the second valve on the output side of the amplification flow path so that the sample solution containing the amplification product is supplied from the amplification section to the detection section in the supply of the first cleaning solution. By connecting to the input side of the detection flow path,
The first, second, and third channels connecting the third and fourth flow paths to the input side of the detection flow path so as to supply the insertion agent solution and the second cleaning solution that do not pass through the amplification unit, respectively. , 4th and 5th channels,
Equipped with
A second plate made of a hard material is provided on the packing, and the packing is pressed and sandwiched between the first plate and the second plate, and the first, second, and second plates are inserted. The 3rd and 4th syringes, the amplification unit, the detection unit, the 1st, 2nd, 3rd, 4th and 5th flow paths and the 1st and 2nd valves are formed by being liquidtightly sealed. Nucleic acid detection cassette having a structure. The third, fourth, fifth and sixth valves connected to the first, second, third and fourth flow paths, respectively.
The third valve allows the sample solution to be supplied to the amplification flow path through the third valve.
The fourth valve allows the supply of the first cleaning liquid to the amplification flow path via the fourth valve.
The fifth valve allows the insertion agent liquid to be supplied to the detection flow path via the fifth valve, and the sixth valve supplies the second cleaning liquid to the detection flow path via the sixth valve. Forgive 3rd, 4th, 5th and 6th valves and
The nucleic acid detection cassette of claim 1 further comprising. The third, fourth, fifth and sixth valves are
The storage of the sample solution, the first cleaning solution, the insertion agent solution and the second cleaning solution in the first, second, third and fourth syringes was maintained.
When the sample solution, the first cleaning solution, the insertion agent solution, and the second cleaning solution are extruded, the pumps are opened to accompany the deformation of the first, second, third, and fourth syringes. The nucleic acid detection cassette according to claim 2, which provides a function. A waste liquid tank formed by a recess provided in the first and second plates and a through groove formed in the packing, and a waste liquid tank.
A sixth flow path formed between the first plate and the packing, the output side of the detection flow path is connected to the waste liquid tank, and the second cleaning liquid is connected to the detection flow path. A sixth flow path that guides the sample solution extruded from the detection unit to the waste liquid tank with the supply of
The nucleic acid detection cassette of claim 1 further comprising. The first, second, third and fourth syringes are provided in the first, second, third and fourth syringe recesses and the packing formed in the first plate, and the first The first, elastically deformable, which covers each of the second, third, and fourth syringe recesses and has a bulging shape that bulges in the direction opposite to the recess so as to define a syringe space between the recesses and the recesses. The first, second, and third in the first, second, third, and fourth syringe grooves formed by the second, third, and fourth bulges and provided on the second plate. The nucleic acid detection cassette according to claim 1, wherein a fourth bulge and a fourth bulge are arranged respectively and deformed from the outside through the first, second, third and fourth syringe grooves. The plurality of electrodes further include a counter electrode for applying a voltage to the working electrode and a reference electrode for detecting the reference voltage, and the electrode pad is connected to the working electrode, the counter electrode, and the reference electrode. The nucleic acid detection cassette according to claim 1. In the liquid feeding method in the nucleic acid detection cassette according to claim 1.
The first syringe is crushed, and the sample solution is supplied from the first syringe to the amplification section via the first flow path.
With the first and second valves closed, the amplification unit is heated from the outside to amplify the nucleic acid in the sample solution to produce an amplification product.
The second syringe is crushed, and the first cleaning solution is supplied from the second syringe to the amplification section via the second flow path to obtain the sample solution containing the amplification product in the amplification section. It is supplied to the detection unit via the fifth flow path, and is supplied to the detection unit.
A hybridization reaction is generated in the detection unit to cause a hybridization reaction.
The fourth syringe is crushed, the second cleaning solution is supplied from the fourth syringe to the detection unit via the fourth flow path, and the sample solution is pushed out of the detection unit.
The third syringe is crushed, the insertion agent liquid is supplied from the third syringe to the detection unit via the third flow path, and the second cleaning liquid is pushed out of the detection unit.
A method of sending a liquid in a nucleic acid detection cassette. In the liquid feeding method in the nucleic acid detection cassette according to claim 2.
The third valve is opened and the sample solution is supplied to the amplification unit via the third valve and the first valve, and after the fourth valve is opened, the first cleaning liquid is added to the fourth valve. The sample solution containing the amplification product is supplied from the amplification section to the amplification section via the valve and the first valve, and the sample solution containing the amplification product is supplied from the amplification section to the amplification section via the second valve. The second cleaning liquid is supplied to the detection flow path without being pushed out into the flow path and supplied to the detection section, and the sixth valve is opened and does not pass through the amplification section, and the sample solution is the detection. It is pushed out of the detection unit via the output side of the flow path, and then the second cleaning liquid in the detection flow path is pushed out by the supply of the insertion agent liquid via the fifth valve that does not pass through the amplification unit. ,
A method of sending a liquid in a nucleic acid detection cassette.

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