JP6081113B2 - Nucleic acid detection device and nucleic acid detection apparatus - Google Patents

Description

  Embodiments described herein relate generally to a nucleic acid detection device and a nucleic acid detection apparatus that detect nucleic acids using electrical signals.

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

  As one of devices for detecting a nucleic acid, a device using a DNA chip can be given. A DNA chip is a device in which a plurality of nucleic acid probes are immobilized on a substrate, and is characterized in that a large number of nucleic acid sequences can be detected at one time. In general, a nucleic acid probe is immobilized on a sensor surface by being dropped onto each sensor in a state dissolved in a liquid. Since different nucleic acid probes are immobilized on each sensor, it is necessary to avoid contact of each droplet. Thus, there is a limitation that the distance between each sensor must be longer than the diameter of the nucleic acid probe droplet. High integration of sensors and reduction in device size are indispensable for cost reduction.

  On the other hand, a device called μ-TAS capable of sequentially performing a plurality of reactions involving a plurality of reagents in one device has been actively researched and developed. These are characterized by comprising a reagent holding region, a nucleic acid reaction region (amplification region), a sensor region, and the like, and having a flow path connecting them.

  Furthermore, a nucleic acid detection apparatus for detecting a nucleic acid using a nucleic acid detection device incorporating the above-described current detection type DNA chip has been developed. When nucleic acid detection is performed in such a device, it is necessary to perform a plurality of reactions using a plurality of reagents. For example, nucleic acid extraction reaction, nucleic acid purification reaction, nucleic acid amplification reaction, nucleic acid hybridization reaction and the like. Since these reagents for nucleic acid detection are generally expensive, it is desirable to reduce the amount used as much as possible. Therefore, a nucleic acid detection device that can efficiently supply a reagent to each sensor by forming a flow channel on the device and arranging each sensor in the flow channel has been developed.

US Pat. No. 5,776,672 US Pat. No. 5,972,692 US Pat. No. 6,294,670 JP 2004-61427 A Japanese Patent No. 4127679 JP 2008-263959 A

  In order to reduce the cost of nucleic acid detection devices, miniaturization of nucleic acid detection devices is required. In addition, miniaturization of a nucleic acid detection device requires that each member be highly integrated. However, the interval between the reaction regions of the nucleic acid in the flow channel formed in the flow channel forming member depends on the diameter of the droplet of the nucleic acid probe dropped in each region, and thus cannot be narrowed. Therefore, the flow path forming member must highly integrate the formed flow path shape after satisfying the above-described requirements.

  Further, in general, the flow path forming member is manufactured by molding using a mold in forming the flow path. However, if the flow path shape is highly integrated, for example, the interval between adjacent flow paths is narrowed, it becomes difficult to manufacture a mold for the flow path forming member.

  Furthermore, in a nucleic acid detection device that uses a sensor for nucleic acid detection, the distance between the sensors depends on the diameter of the droplet of the nucleic acid probe dropped onto each sensor. Further, each sensor is wired on the substrate and each pad for outputting the output of each sensor to the nucleic acid detection device. For this reason, when the sensor is highly integrated, it becomes difficult to route the wiring.

  An object of the present invention is to provide a highly integrated nucleic acid detection device and nucleic acid detection apparatus.

According to the embodiment,
A substrate having a substrate surface ;
So as to form a plurality of rows including a first row and a second row adjacent to the first row, a plurality of sensor units arranged on the substrate surface along a first direction, the plurality of sensor units Includes a first sensor portion arranged along the first row and a second sensor portion arranged along the second row, and the first and second sensor portions do not face each other. a plurality of sensor units that have no staggered arrangement are staggered along the direction,
A first member comprising:
A second member having a facing surface facing the substrate surface and having a groove portion defining a flow path for feeding a reagent on the plurality of sensor portions formed on the facing surface; The plurality of second members are stacked on the first member so that the groove portion accommodates the plurality of sensor portions, and the groove portions are arranged along the first direction corresponding to the sensor portions . A second member including a groove portion of the row and a folded portion connecting the groove portions ;
There is provided a nucleic acid detection device for detecting a nucleic acid by an electric signal from the sensor unit comprising:
Moreover, according to the embodiment,
A substrate having a substrate surface ;
So as to form a plurality of rows including a first row and a second row adjacent to the first row, a plurality of sensor units arranged on the substrate surface along a first direction, the plurality of sensor units Includes a first sensor portion arranged along the first row and a second sensor portion arranged along the second row, and the first and second sensor portions do not face each other. a plurality of sensor units that have no staggered arrangement are staggered along the direction,
A first member comprising:
A second member having a facing surface facing the substrate surface and having a groove portion defining a flow path for feeding a reagent on the plurality of sensor portions formed on the facing surface;
The plurality of second members are stacked on the first member so that the groove portion accommodates the plurality of sensor portions, and the groove portions are arranged along the first direction corresponding to the sensor portions . Including a groove portion of the row and a folded portion connecting the groove portions ;
The groove portion includes a plurality of first groove regions provided opposite to the sensor unit and a second groove region connecting the first groove regions adjacent to each other, and the width of the first groove region . Is a second member formed wider than the width of the second groove region ,
There is provided a nucleic acid detection device for detecting a nucleic acid by an electric signal from the sensor unit comprising:

1 is a perspective view showing a schematic configuration of a nucleic acid detection device according to an embodiment. The top view of the device for nucleic acid detection concerning an embodiment. The figure which shows schematic structure of the nucleic acid detection apparatus which concerns on embodiment. Sectional drawing of the device for nucleic acid detection which concerns on embodiment. The top view of the DNA chip concerning an embodiment. The top view which shows the positional relationship of the sensor part which concerns on embodiment. The top view which shows the other example of the positional relationship of the sensor part which concerns on embodiment. The top view used as an example of the groove part which concerns on embodiment.

  Various embodiments will be described below with reference to the drawings. In addition, the same code | symbol shall be attached | subjected to a common structure through embodiment, and the overlapping description is abbreviate | omitted. In addition, each drawing is a schematic diagram for promoting the embodiment and its understanding, and its shape, dimensions, ratio, etc. are different from the actual device, but these are considered in consideration of the following description and known techniques. The design can be changed as appropriate.

  FIG. 1 is a perspective view showing a schematic configuration as an example of a nucleic acid detection device (nucleic acid detection cassette) 10 according to an embodiment. FIG. 2 is a top view illustrating an example of the nucleic acid detection device 10 according to the embodiment. The nucleic acid detection device 10 includes elements of a flow path packing 11, an upper plate 12, a lower plate 13, and a DNA chip 14. The nucleic acid detection device 10 has a substantially rectangular shape configured by sandwiching a flow path packing 11 and a DNA chip 14 between an upper plate 12 and a lower plate 13. Note that the long side of the nucleic acid detection device 10 corresponds to the X axis (also referred to as the X direction), and the short side orthogonal to the long side corresponds to the Y axis (also referred to as the Y direction). The DNA chip 14 is sandwiched between the flow path packing 11 and the lower plate 13. The direction in which the lower plate 13, the DNA chip 14, the flow path packing 11, and the upper plate 12 are stacked in this order is referred to as the stacking direction of the nucleic acid detection device 10. The stacking direction perpendicular to the long side and the short side corresponds to the Z axis. Furthermore, the outer surface of the upper plate 12 in the stacking direction of the nucleic acid detection device 10 is referred to as the surface of the nucleic acid detection device 10. Similarly, the outer surface of the lower plate 13 in the stacking direction of the nucleic acid detection device 10 is referred to as the back surface of the nucleic acid detection device 10.

  The flow path packing 11 is made of a thin member. The flow path packing 11 is comprised by soft materials (elastic material), such as silicone and an elastomer, for example. In the flow path packing 11, elements included in the flow path packing 11 are integrally formed. A surface of the flow path packing 11 that faces (opposes) the upper plate 12 is referred to as a surface of the flow path packing 11. Similarly, the surface of the flow path packing 11 that faces the lower plate 13 or the DNA chip 14 is referred to as the back surface of the flow path packing 11. The flow path packing 11 includes an inlet member 111a, an inlet member 111b, an inlet member 111c. A sample syringe 112a, a cleaning syringe 112b, an insertion agent syringe 112c, a flow path forming member 113, a waste liquid syringe 114, a check valve member 115a, a check valve member 115b, and a check valve member 115c are provided.

  The inlet member 111a includes an opening for injecting a specimen in order to fill the nucleic acid detection device 10 with a liquid specimen (also referred to as a nucleic acid sample). The opening of the inlet member 111 a is exposed on the surface of the nucleic acid detection device 10 without being covered by the upper plate 12. The inlet member 111b includes an opening for injecting a cleaning liquid in order to fill the nucleic acid detection device 10 with a cleaning liquid (also referred to as a cleaning reagent). The opening of the inlet member 111b is exposed on the surface of the nucleic acid detection device 10 without being covered by the upper plate 12. The inlet member 111c is. In order to fill the nucleic acid detection device 10 with an insertion agent for redox reaction, an opening for injecting an insertion agent (also referred to as a detection reagent) is provided. The opening of the inlet member 111 c is exposed on the surface of the nucleic acid detection device 10 without being covered by the upper plate 12.

  The sample syringe 112a has a container shape. The sample syringe 112a stores a sample to be injected through the injection port member 111a. The cleaning syringe 112b has a container shape. The cleaning syringe 112b stores the cleaning liquid that is injected through the inlet member 111b. The insertion agent syringe 112c is configured in a container shape. The insertion agent syringe 112c stores the insertion agent injected through the inlet member 111c.

The flow path forming member 113 is a member facing the DNA chip 14 described later. The flow path forming member 113 includes a groove 113a on the back surface. The groove 113a is provided from the inlet 113b to the outlet 113c. The groove 113a is formed on the back surface of the flow path forming member 113 facing a DNA chip 14 (the surface of the substrate 141) described later. The groove 113a is opposed to each sensor unit 142 provided on the DNA chip 14 described later. The groove portion 113a has a shape along the arrangement of the sensor portions 142. That is, the groove portions 113a are formed in a plurality of rows along the X axis so as to face the sensor portions 142. Furthermore, the groove 113a functions as a flow path for feeding a reagent onto each sensor unit 142. That is, the groove 113a defines a region for processing a nucleic acid extraction reaction, a nucleic acid purification reaction, a nucleic acid amplification reaction, a nucleic acid hybridization reaction, nucleic acid detection, and the like. The groove 113a is formed in the flow path forming member 113 so as to include substantially straight portions along one direction (for example, the X axis) in four rows in parallel, and further include three folded portions that connect them. In addition, the groove part 113a should just be provided with two or more substantially linear parts along the X-axis in parallel. In the inlet 113b, the sample flows from the sample syringe 112a, the cleaning liquid flows from the cleaning syringe 112b, and the insertion agent flows from the insertion agent syringe 112c.
The waste liquid syringe 108 is configured in a container shape. The waste liquid syringe 108 stores the liquid flowing out from the discharge port 113 c of the flow path forming member 113.
The check valve member 115a is provided at a predetermined position in the flow path connecting the sample syringe 112a and the groove 113a. The valve member 115a prevents the sample from flowing back into the sample syringe 112a. The check valve member 115b is provided at a predetermined position in the flow path connecting the cleaning syringe 112b and the groove 113a. The check valve member 115b prevents the cleaning liquid from flowing back into the cleaning syringe 112b. The check valve member 115c is provided at a predetermined position in the flow path connecting the insertion agent syringe 112c and the groove 113a. The check valve member 115c prevents the insertion agent from flowing back into the insertion agent syringe 112c.

  The upper plate 12 is composed of a thin member. The upper plate 12 is made of a material harder than the flow path packing 11. The upper plate 12 is made of a hard material such as plastic, glass, or metal. The upper plate 12 is in close contact with the flow path packing 11 or the DNA chip 14. That is, the upper plate 12 is used for sealing the flow path packing 11 and the DNA chip 14. The upper plate 12 includes an opening 121a, an opening 121b, an opening 121c, an opening 122, and an opening 123. The opening 121a, the opening 121b, and the opening 121c are provided at positions opposed to the inlet member 111a, the inlet member 111b, and the inlet member 111c, respectively. The opening 121a, the opening 121b, and the opening 121c are configured so that the inlet member 111a, the inlet member 111b, and the inlet member 111c can pass therethrough.

  The opening 122 is provided at a position opposite to an electrode pad region 1431 configured by a plurality of electrode pads 143 provided (arranged) in the DNA chip 14 described later. The opening 122 is provided in substantially the same shape as the electrode pad region 1431. Similarly, the opening 123 is provided at a position opposite to an electrode pad region 1441 configured by a plurality of electrode pads 144 provided (arranged) in the DNA chip 14 described later. The opening 122 is provided in substantially the same shape as the electrode pad region 1441.

  The lower plate 13 is composed of a thin member. The lower plate 13 is made of a material harder than the flow path packing 11. The lower plate 13 is made of a hard material such as plastic, glass, or metal. The lower plate 13 is in close contact with the flow path packing 11 or the DNA chip 14. That is, the lower plate 13 is used for sealing the flow path packing 11 and the DNA chip 14. The lower plate 13 has an opening at a position facing a region in the vicinity of each sensor unit 142 provided (arranged) on the DNA chip 14 described later, that is, a region of the DNA chip 14 defined as a flow path by the groove 113a. 131 (see FIG. 4).

The DNA chip 14 includes a substrate 141, a plurality of sensor units 142, a plurality of electrode pads 143, and a plurality of electrode pads 144. The surface of the DNA chip 14 (substrate 141) facing the upper plate 12 is referred to as the surface of the DNA chip 14. Similarly, the surface of the DNA chip 14 (substrate 141) facing the lower plate 13 is referred to as the back surface of the DNA chip 14.
The substrate 141 is formed in a substantially rectangular shape having two sides along the X axis and two sides along the Y axis, and the sensor part 142, the electrode pad 143, and the electrode pad 144 are provided on the surface.
The sensor unit 142 is provided at a substantially central portion of the Y axis on the surface of the DNA chip 14. The sensor unit 142 is a sensor (electrode) made of a conductive member. In the sensor unit 142, various nucleic acid probes for detecting a target nucleic acid are immobilized, and the target nucleic acid is detected. In addition, although the one sensor part 142 is comprised by one sensor in FIG. 3, you may be comprised by two or more sensors. The sensors constituting each sensor unit 142 are, for example, circular with substantially the same diameter. In addition, the sensor which comprises each sensor part 142 may be a rectangle. The sensor portions 142 are arranged on the substrate 141 in four substantially linear shapes along one direction so as to face the groove portions 113a, and a plurality of sensor portions 142 are arranged per row. Here, each sensor part 142 is demonstrated as what is arrange | positioned along the X-axis. The positional relationship of each sensor unit 142 will be described later. In addition, the area | region of each sensor part 142 vicinity on the surface of the board | substrate 141, ie, the area | region of the board | substrate 141 prescribed | regulated as a flow path by the groove part 113a of the flow path formation member 113 on the surface of the board | substrate 141 is called sensor area | region 1421. To do. Note that the sensor region 1421 is also referred to as a flow channel region because it corresponds to a region defined as a flow channel by the groove 113a. The sensor units 142 may be arranged on the substrate 141 in a plurality of rows (even rows, odd rows) other than 4 rows along the X axis in a substantially straight line, and a plurality of sensors may be arranged per row. In this case, the substantially straight line portion of the groove 113a is formed in the flow path forming member 113 along the X axis in an even or odd number of rows corresponding to the number of rows of the sensor portion 142.

  The electrode pad 143 is provided on the surface of the substrate 141 so as to face the opening 122 of the upper plate 12. A plurality of electrode pads 143 are provided along the first side along the X axis of the substrate 141. The electrode pads 143 are arranged in a plurality of rows substantially along the first side over substantially the whole or a part of the first side, and a plurality of electrode pads 143 are arranged per row. The electrode pad 143 is made of a conductive member. The electrode pad 143 is for taking out the detection signal of the sensor unit 142 and transmitting it to the nucleic acid detection device 20 described later. One electrode pad 143 is connected to one sensor unit 142 by wiring (not shown) provided on the substrate 141. Note that a region where the electrode pad 143 is provided on the surface of the substrate 141 is referred to as a first pad region 1431. That is, the first pad region 1431 includes all of the plurality of electrode pads 143 arranged, and is defined by connecting the outer edges of the electrode pads 143 arranged outside of all of the plurality of electrode pads 143 arranged. It is an area to be done. Note that although the electrode pad 143 is provided so that the first pad region 1431 has a substantially rectangular shape, the present invention is not limited to this. Similarly, the electrode pad 144 is provided on the surface of the substrate 141 at a position different from the electrode pad 143 so as to face the opening 123 of the upper plate 12. A plurality of electrode pads 144 are provided along a substantially parallel second side opposite to the first side along the X axis of the substrate 141. The electrode pads 144 are arranged in a plurality of rows substantially along the second side over substantially the whole or part of the second side, and a plurality of electrode pads 144 are arranged per row. The electrode pad 144 is made of a conductive member. The electrode pad 144 is for taking out the detection signal of the sensor unit 142 and transmitting it to the nucleic acid detection device 20 described later. Note that one electrode pad 144 is connected to one sensor unit 142 by wiring (not shown) provided on the substrate 141. Note that a region where the electrode pad 144 is provided on the surface of the substrate 141 is referred to as a second pad region 1441. That is, the second pad region 1441 includes all of the plurality of electrode pads 144 arranged, and is defined by connecting the outer edges of the electrode pads 144 arranged outside of all of the plurality of electrode pads 144 arranged. It is an area to be done. Note that although the electrode pad 144 is provided so that the second pad region 1441 has a substantially rectangular shape, the present invention is not limited to this.

A flow path connecting the injection port member 111a and the sample syringe 112a, a flow path connecting the injection port member 111b and the cleaning syringe 112b, a flow path connecting the injection port member 111c and the insertion syringe 112c, the sample syringe 112a, and washing The flow path connecting the syringe 112b, the insertion syringe 112c, and the groove 113a, and the flow path connecting the groove 113a and the waste liquid syringe 114 are formed by any one of the flow path packing 11, the upper plate 12, and the lower plate 13. Alternatively, it may be formed by a combination of at least two of the flow path packing 11, the upper plate 12, and the lower plate 13.
With the above-described configuration, the nucleic acid detection device 10 can perform a process from nucleic acid amplification reaction to nucleic acid detection in a flow path (sensor region 1421) formed by the groove 113a and the substrate 141.

  Next, the structure of the nucleic acid detection apparatus 20 for detecting a nucleic acid using the nucleic acid detection device 10 will be described. FIG. 3 is a diagram illustrating a schematic configuration of the nucleic acid detection device 20 according to the embodiment. 4 is a cross-sectional view of the nucleic acid detection device 10 taken along the line AA in FIG. FIG. 4 also shows elements constituting the nucleic acid detection device 20 that contacts the nucleic acid detection device 10 inserted in the nucleic acid detection device 20. In the nucleic acid detection apparatus 20, the nucleic acid detection device 10 is inserted along the X axis. That is, the groove portions 113a (the sensor portion 142 and the sensor region 1421 are the same) are provided in a plurality of rows along the insertion direction of the nucleic acid detection device 10 into the nucleic acid detection device 20. The nucleic acid detection device 20 includes a connector 201, a connector 202, and a temperature control unit 203.

  The connector 201 is provided at a position facing the opening 122 when the nucleic acid detection cassette 10 is inserted into the nucleic acid detection device 20. The connector 201 has a size that can be inserted into and removed from the opening 122. The connector 201 comes into contact with the electrode pads 143 in the electrode pad region 1431 through the connector pins 2011 for current measurement. Similarly, the connector 202 is provided at a position facing the opening 123 when the nucleic acid detection cassette 10 is inserted into the nucleic acid detection apparatus 20. The connector 202 has a size that can be inserted into and removed from the opening 123. The connector 202 comes into contact with each electrode pad 144 in the electrode pad region 1441 through the connector pin 2021 for current measurement. The connector 201 and the connector 202 take out detection signals from the electrode pad 143 and the electrode pad 144, respectively. The nucleic acid detection device 20 performs nucleic acid detection based on detection signals taken out via the wirings 2012 and 2022 connected to the connector 201 and the connector 202, respectively, and determines the presence or absence of the target nucleic acid.

  The temperature control unit 202 is provided at a position facing the opening 131 provided in the lower plate 13 when the nucleic acid detection cassette 10 is inserted into the nucleic acid detection device 20.

The temperature control unit 202 has a size that can be inserted into and removed from the opening 131. The temperature control unit 202 comes into contact with the back surface of the substrate 141 facing the sensor region 1421. The temperature control unit 202 optimally controls the flow path in the groove 113a, that is, the temperature (liquid temperature) of the sensor region 1421 from the back side of the substrate 141.

  FIG. 5 is a top view illustrating an example of the DNA chip 14 on which the flow path forming members 113 according to the embodiment are stacked. FIG. 6 is a plan view showing the positional relationship of the sensor unit 142 arranged on the substrate 141. With reference to FIGS. 5 and 6, the positional relationship of each sensor unit 142 and the shape of the groove 113a facing each sensor unit 142 will be described. The sensor unit 142 includes a single sensor.

  The sensor unit 142a is an arbitrary one arranged so that the center of the sensor unit 142a (that is, the center of one sensor constituting the sensor unit 142a) is positioned on an arbitrary column (referred to as a first column) along the X axis. This is a sensor unit 142. The sensor part 142b is adjacent to the sensor part 142a in the first row, and is arranged so that the center of the sensor part 142b (that is, the center of one sensor constituting the sensor part 142b) is positioned on the first row. 142a is a sensor unit 142 adjacent to 142a. The sensor unit 142c is arranged so that the center of the sensor unit 142c (that is, the center of one sensor constituting the sensor unit 142c) is positioned on the first column and one column adjacent to the Y axis (referred to as the second column). This is a sensor unit 142.

  The sensor unit 142c is located between the X-axis position of the outer edge of the sensor unit 142a closest to the sensor unit 142b side and the X-axis position of the outer edge of the sensor unit 142b closest to the sensor unit 142a side in the X-axis. Are arranged as follows. Furthermore, the sensor part 142c is arrange | positioned in the position away from the 1st row | line side rather than the position of the outer edge of the sensor part 142a nearest to the 2nd row | line | column side and the outer edge of the sensor part 142a in the Y-axis. In addition, the relationship between the sensor unit 142 and the sensor unit 142c arranged on the second column and the first column (referred to as the third column) opposite to the first column adjacent to the Y-axis is arranged in the first column described above. Since the relationship between the sensor units 142a and 142b and the sensor unit 142c is the same, the description thereof is omitted.

  As described above, the sensor unit 142c, the sensor unit 142a, and the sensor unit 142b are arranged so as not to be partially opposed in the X axis and the Y axis. Note that, as described above, the sensor unit 142c, the sensor unit 142a, and the sensor unit 142b may be arranged so as not to be partially opposed at least in the Y-axis direction of the X-axis and the Y-axis. That is, the sensor unit 142 arranged in the first row and the sensor unit 142 arranged in the second row adjacent to the first row and the Y-axis are partially in at least the Y-axis direction of the X-axis and the Y-axis. Are arranged alternately in the first row and the second row so as not to be opposed to each other. In the present embodiment, the arrangement of the sensor units 142 as described above is included in the definition of the staggered arrangement.

  According to the arrangement relationship of the sensor unit 142 as described above, the center of the sensor unit 142a, the center of the sensor unit 142b, and the center of the sensor unit 142c form an equilateral triangle or an isosceles triangle with the center of the sensor unit 142c as a vertex. Are arranged to be. Therefore, the distance between the sensor part 142a and the sensor part 142c and the distance between the sensor part 142b and the sensor part 142c can be designed to be longer than the diameter of the droplet of the nucleic acid probe dropped on each sensor part. Therefore, the DNA chip 14 can realize high integration of the sensor unit without increasing its size. The sensor unit 142c is preferably arranged so that the apex angle θ1 of the sensor unit 142c is less than 60 degrees. The reason is as follows. The sensor part 142 a and the sensor part 142 b are coupled by a flow path, but the sensor part 142 c is separated by a flow path forming member 113. Since the cost can be reduced by reducing the amount of the reagent that fills the flow path portion that couples the sensor part 142a and the sensor part 142b, the flow path needs to be as short as possible. On the other hand, the sensor unit 142c does not increase the necessary reagent amount even when the sensor unit 142a and the sensor unit 142b are separated from each other. Therefore, in order to design the sensor unit 142a and the sensor unit 142b as close as possible, and then the sensor unit 142c, it is preferable that θ1 is less than 60 degrees.

  A preferred arrangement example of the sensor units 142c arranged in the second row will be described. Here, a is a distance between the center of the sensor unit 142a and the center of the sensor unit 142b arranged in the first row. The sensor unit 142c arranged in the second row satisfies the above conditions, and the X-axis with respect to the center line between the center of the sensor unit 142a and the center of the sensor unit 142b arranged in the first row. It is preferable that the sensor portion 142c is disposed so that the center thereof is positioned within a range of 25% on the left and right. That is, the center of the sensor unit 142c is positioned within the range of b = 1/2 * a along the X axis with the center line between the center of the sensor unit 142a and the center of the sensor unit 142b as the center. Preferably they are arranged.

  Next, another example of the positional relationship of each sensor unit 142 will be described. FIG. 7 is a plan view showing the positional relationship of the sensor unit 142 arranged on the substrate 141. The sensor unit 142 includes three sensors. Here, an example in which the sensor unit 142 includes three sensors will be described, but the same applies to the case where the sensor unit 142 includes two or more sensors.

  The sensor unit 142d is an arbitrary sensor unit 142 disposed so that the center of each sensor constituting the sensor unit 142d is positioned on a certain row (referred to as a first row) along the X axis. The sensor unit 142e is adjacent to the sensor unit 142d in the first row, and is arranged so that the center of each sensor constituting the sensor unit 142e is positioned on the first row, and is adjacent to the sensor unit 142d. . The sensor unit 142f is a sensor unit 142 arranged so that the center of each sensor constituting the sensor unit 142f is positioned on a first row and a row adjacent to the Y axis (referred to as a second row).

  The sensor unit 142f has an X-axis position on the outer edge of the sensor constituting the sensor unit 142d closest to the sensor unit 142e side in the X axis, and an X of the outer edge of the sensor constituting the sensor unit 142e closest to the sensor unit 142d side. It arrange | positions so that it may fit between the position of an axis | shaft. Further, the sensor unit 142f is arranged at a position farther from the first column side than the Y-axis position of the sensor unit 142d and the sensor unit 142e closest to the second column side on the Y axis. The relationship between the sensor unit 142 and the sensor unit 142f disposed on the second column and the first column adjacent to the Y axis (referred to as the third column) opposite to the first column is the first column described above. Since the relationship between the sensor units 142d and 142e and the sensor unit 142f is the same, the description thereof is omitted.

  As described above, the sensor that constitutes the sensor unit 142f, the sensor that constitutes the sensor unit 142d, and the sensor that constitutes the sensor unit 142e are arranged so as not to be partially opposed in the X axis and the Y axis. As described above, a part of the sensor that constitutes the sensor unit 142f, the sensor that constitutes the sensor unit 142d, and the sensor that constitutes the sensor unit 142e are partially relative to each other in at least the Y-axis direction of the X-axis and the Y-axis. It may be arranged so as not to. That is, the sensor constituting the sensor unit 142 arranged in the first row and the sensor constituting the sensor unit 142 arranged in the second row adjacent to the first row and the Y axis are the X axis and the Y axis. Of these, at least in the Y-axis direction, they are staggered so as not to be partially opposed.

  According to the arrangement relationship of the sensor unit 142 as described above, the center of the sensor unit 142d (the center between the centers of the two outermost sensors in the X axis among the sensors constituting the sensor unit 142d) and the center of the sensor unit 142e. (Center between the centers of the two outermost sensors on the X axis among the sensors constituting the sensor unit 142e), the center of the sensor unit 142f (the two outermost sensors on the X axis among the sensors constituting the sensor unit 142f) Are arranged so as to form an equilateral triangle or an isosceles triangle having the center of the sensor portion 142f as a vertex. Therefore, the distance between the sensor part 142d and the sensor part 142f and the distance between the sensor part 142e and the sensor part 142f can be designed to be longer than the diameter of the droplet of the nucleic acid probe dropped on each sensor part. Therefore, the DNA chip 14 can realize high integration of the sensor unit without increasing its size. In addition, it is preferable that the sensor part 142f is arrange | positioned so that the apex angle (theta) 2 of the sensor part 142f may be less than 60 degree | times similarly to the example shown in FIG.

  A preferred arrangement example of the sensor units 142f arranged in the second row will be described. Here, the distance between the center of the sensor unit 142d arranged in the first row and the center of the sensor unit 142e is a ′. The sensor part 142f arranged in the second row satisfies the above conditions, and has an X axis with respect to a center line between the center of the sensor part 142d and the center of the sensor part 142e arranged in the first row. It is preferable that the sensor portion 142f is arranged so that the center thereof is located within a range of 25% on the left and right. That is, the center of the sensor unit 142f is located within the range of b ′ = 1/2 * a ′ along the X axis with the center line between the center of the sensor unit 142d and the center of the sensor unit 142e as the center. It is preferable that they are arranged as described above.

Next, the shape of the groove 113a will be described. As shown in FIG. 5, the groove 113a has a first groove 113d (corresponding to a nucleic acid reaction region) facing the vicinity of the sensor 114 and a second groove connecting the first groove 113d in a substantially linear portion. 113e. The width of the first groove 113d (the width in the direction (Y-axis direction) orthogonal to the direction (X-axis direction) in which the groove 113a is formed) is wider than the width of the second groove 113e. That is, the width of the flow channel region defined by the first groove 113d is wider than the width of the flow channel region defined by the second groove 113e. The flow path area defined by the first groove 113d is substantially circular. The groove 113a is formed in the flow path forming member 113 in a constricted shape by the first groove 113f and the second groove 113e. Further, with the staggered arrangement of the sensor portions 142, the first groove portions 113d are formed in the flow path forming member 113 in a staggered arrangement so as to face the vicinity of the sensor portion 114. That is, the first groove 113d formed in a certain row (referred to as the first row) and the first groove 113d arranged in the second row adjacent to the first row and the Y axis are the X axis and the Y axis. Are formed in the flow path forming member 113 so as to be staggered in the first row and the second row so as not to be partially opposed at least in the Y-axis direction. In the present embodiment, the arrangement of the first groove portions 113d formed in the flow path forming member 113 as described above is included in the definition of the staggered arrangement. According to the staggered arrangement of the first grooves 113d, in the grooves 113a, the distance between the first row and the second row is substantially the same along the X axis. In addition, since the preferable arrangement | positioning relationship of the 1st groove part 113d is the same as the preferable arrangement | positioning relationship of the above-mentioned sensor part 142, description is abbreviate | omitted.
The first groove 113d is not limited to the shape shown in FIG. 5, and may be formed in the flow path forming member 113 in another shape. For example, as shown in FIG. 8, the first groove 113d may be formed in the flow path forming member 113 so that the flow path area defined by the first groove 113d is substantially rectangular (rectangular). .
The groove 113a is not constricted, and the width of the first groove 113d and the width of the second groove 113e are substantially the same, that is, the substantially linear portion along the arrangement direction of the sensor 142 has a Z-axis cross-sectional area. The flow path forming member 113 may be formed in a straight line so as to be the same.
In the present embodiment, the sensor unit 142 is disposed along the X axis, and the groove 113a is formed in the flow path forming member 113 along the X axis. However, the present embodiment is not limited thereto. The sensor part 142 may be arranged along the Y axis, and the groove part 113a may also be formed in the flow path forming member 113 along the Y axis. The arrangement direction of the sensor part 142 and the groove part 113a in the flow path forming member 113 may be The formation direction may be substantially the same.
In addition, the flow path forming member 113 in which the groove portions 113 are formed in a constricted shape and a staggered arrangement is not only a current detection type nucleic acid detection device using the sensor unit 142 but also a nucleic acid detection device that does not use the sensor unit 142. Applicable.

  According to the present embodiment, the first groove portions 113d are formed in the flow path forming member 113 in a staggered arrangement, so that the flow path forming members 113 are formed in the adjacent rows. High integration can be achieved while keeping the distance of Since the first groove 113d is formed in the flow path forming member 113 in a staggered arrangement, the interval between adjacent rows can be made substantially constant and wide, so that the mold for the flow path forming member 113 can be easily manufactured. Become. These characteristics relate to all nucleic acid detection devices that use the flow path forming member 113 in which a flow path is formed. Furthermore, by arranging the sensor portions 142 on the substrate 141 in a staggered manner, the sensor can be more highly integrated than before, and the nucleic acid detection device can be downsized. Furthermore, by providing the sensor portions 142 in a staggered arrangement on the substrate 141, it is easy to route the wiring connecting the sensor portion 142 and the pad portion 143 or the pad portion 144. For example, since the wiring can be straightened from all the sensor units 142 to the pad unit 143 or the pad unit 144, the strength of the wiring is increased. This feature relates to a nucleic acid detection device that uses the sensor unit 142 in addition to the flow path forming member 113 in which the flow path is formed.

Example 1
A specific example of nucleic acid detection using the nucleic acid detection device 10 and the nucleic acid detection apparatus 20 according to the above-described embodiment will be described below.

1. Preparing a cassette with a built-in nucleic acid detection device
1-1. Preparation of Nucleic Acid Detection Device Five types of nucleic acid probes (sequences A to A) shown in Table 1 below are applied to each electrode on the nucleic acid detection device 10 (hereinafter, corresponding to a sensor constituting each sensor unit 142). E) was immobilized. Immobilization was carried out by dropping a solution containing each nucleic acid probe onto each electrode set, and then washing and removing excess nucleic acid probes. In addition, the following 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 20th electrode groups constitute separate sensor parts 142, respectively. .

1) Negative control ... 1 and 2 electrodes 2) HPV-A detection ... 3 and 4 electrodes 3) HPV-B detection ... 5 and 6 electrodes 4) HPV-C detection ... 7 and 8 electrodes 5) For detecting HPV-D ... No.9,10 electrode

1-2. Assembly of Nucleic Acid Detection Device The nucleic acid detection device 10 was configured by attaching the flow path packing 11 including the flow path forming member 113 capable of forming a reaction region on the DNA chip 14. An inlet 113b is formed in the flow path forming member 113, and the flow path forming member 113 is fixed to the DNA chip 14 so that liquid does not leak.
The cleaning reagent is SSC, and the detection reagent is Hoechst 33258.

2. Nucleic acid detection using nucleic acid detection device 10
First, a nucleic acid sample having an amplified HPV-B sequence was sent to the groove 113a and held at 45 ° C. for 10 minutes to perform a hybridization reaction with the nucleic acid detection probe on the DNA chip 14. Non-specifically adsorbed nucleic acid was removed by feeding the cleaning reagent to the groove 113a and holding it at 30 ° C. for 5 minutes. The detection reagent was fed to the groove 113a and kept at room temperature for 3 minutes to cause the detection reagent to react with the nucleic acid. Finally, nucleic acid detection was performed by measuring the current value obtained from each electrode of the DNA chip 14.

3. result
The electrode on which the HPV-B detection probe was immobilized had a significantly larger current value compared to the other electrodes, so that the HPV-B sequence in the sample was detected normally. I understood.

  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 novel 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 scope and gist of the invention, and are included in the invention described in the claims and the equivalents thereof.

  DESCRIPTION OF SYMBOLS 10 ... Device for nucleic acid detection, 11 ... Flow path packing, 12 ... Upper plate, 13 ... Lower plate, 14 ... DNA chip, 20 ... Nucleic acid detection apparatus, 111a ... Injection port member, 111a ... Injection port member, 111c ... Injection port Element. 112a ... specimen syringe, 112b ... washing syringe, 112c ... insertion syringe, 113 ... channel forming member, 113a ... groove, 113b ... inlet, 113c ... discharge port 113c, 113d ... first groove, 113e ... second Groove 114, waste syringe, 115a ... check valve member, 115b ... check valve member, 115c ... check valve member, 121a ... opening, 121b ... opening, 121c ... opening, 122 ... opening, 123 ... Opening portion 131... Opening portion 141... Substrate 142, 142 a, 142 b, 142 c, 142 d, 142 e, 142 f ... Sensor portion 143 ... Electrode pad, 144 ... Electrode pad, 201 ... Connector, 202 ... Connector, 203 ... Temperature Control unit, 1421 ... sensor area (flow path area), 1431 ... pad area, 1441 ... pad area.

Claims (7)

A substrate having a substrate surface ;
So as to form a plurality of rows including a first row and a second row adjacent to the first row, a plurality of sensor units arranged on the substrate surface along a first direction, the plurality of sensor units Includes a first sensor portion arranged along the first row and a second sensor portion arranged along the second row, and the first and second sensor portions do not face each other. a plurality of the sensor unit that has no staggered arrangement are staggered along the direction,
A first member comprising:
A second member having a facing surface facing the substrate surface and having a groove portion defining a flow path for feeding a reagent on the plurality of sensor portions formed on the facing surface; The plurality of second members are stacked on the first member so that the groove portion accommodates the plurality of sensor portions, and the groove portions are arranged along the first direction corresponding to the sensor portions . A second member including a groove portion of the row and a folded portion connecting the groove portions ;
A nucleic acid detection device for detecting a nucleic acid by an electrical signal from the sensor unit comprising: The sensor unit includes a nucleic acid detection device according to claim 1 comprising a nucleic acid probe for immobilized nucleic acid detection on the substrate surface. A substrate having a substrate surface ;
So as to form a plurality of rows including a first row and a second row adjacent to the first row, a plurality of sensor units arranged on the substrate surface along a first direction, the plurality of sensor units Includes a first sensor portion arranged along the first row and a second sensor portion arranged along the second row, and the first and second sensor portions do not face each other. a plurality of sensor units that have no staggered arrangement are staggered along the direction,
A first member comprising:
A second member having a facing surface facing the substrate surface and having a groove portion defining a flow path for feeding a reagent on the plurality of sensor portions formed on the facing surface;
The plurality of second members are stacked on the first member so that the groove portion accommodates the plurality of sensor portions, and the groove portions are arranged along the first direction corresponding to the sensor portions . Including a groove portion of the row and a folded portion connecting the groove portions ;
The groove portion includes a plurality of first groove regions provided opposite to the sensor unit and a second groove region connecting the first groove regions adjacent to each other, and the width of the first groove region . Is a second member formed wider than the width of the second groove region ,
A nucleic acid detection device for detecting a nucleic acid by an electrical signal from the sensor unit comprising: It said first groove region, corresponding to the sensor portion are formed in a staggered arrangement, the device for detecting a nucleic acid of claim 3. The plurality of groove portions include first and second groove portions facing the first and second sensor portions arranged in the first row and the second row, respectively, and the first and second groove portions. Comprises a plurality of first groove regions and a second groove region connecting the first groove regions adjacent to each other ,
The first groove region in the first groove portion is disposed so as not to face each other in the second direction with respect to the first groove region in the second groove portion. The nucleic acid detection device according to claim 3 , wherein the first groove regions in the second groove portion are alternately arranged along the first direction and arranged in a staggered pattern . A plurality of electrode pads arranged along a plurality of rows in the first direction on the substrate surface and connected to each of the plurality of sensor portions via wirings in order to take out detection signals of the sensor portions ; ,
The nucleic acid detection device according to claim 1 or 3, further comprising: The sensor unit includes an electrode made of a conductive member and a target nucleic acid detection probe formed on the electrode, and the nucleic acid detection probe is formed by dropping a solution containing a nucleic acid probe. The nucleic acid detection device according to claim 1 or 3 .

Images