Patent ID: 12257576

DESCRIPTION OF EMBODIMENTS

In the following, embodiments of a biochemical analysis device according to the present invention will be described with reference to the drawings. Note that in order to indicate the orientations in the drawings, XYZ coordinate systems are written in the drawings.

First Embodiment

FIG.1shows the overall structure of a biochemical analysis device. The biochemical analysis device according to the present invention exemplifies a device performing electrophoresis amplifying nucleic acid extracted from a test sample, labeling the nucleic acid, and then reading the base sequence of the nucleic acid. In order to amplify nucleic acid, the nucleic acid mixed with a reagent is kept at a predetermined temperature, or in order to perform electrophoresis, the amplified nucleic acid is supplied to a thin tube called a capillary.

A device main body101is connected to a control computer125with a communication cable. The control computer125accepts an input from an operator, controls the functions of the biochemical analysis device, gives and receives data detected at the device main body101, and displays data that is given and received. The device main body101includes a capillary array114, a pump mechanism103, a thermostat115, the conveyor122, a high-voltage power supply104, a light source111, and an optical detector112. In the following, these components will be described.

The capillary array114is a replacement member composed of one or a plurality (e.g. two to 96) of capillaries102, including a load header124, a detecting unit113, and a capillary head129. At one end of the capillary array114, the load header124is provided to supply a sample into the capillary102, forming a cathode end126to which a negative voltage is applied. At the other end of the capillary array114, a plurality of capillaries102is bundled in one by the capillary head129, and is connected in a pressure-proof secret structure to a gel block106. The detecting unit113, to which laser light is applied, is provided between the load header124and the capillary head129.

The capillary102is a glass tube having an inner diameter a few tens to a few hundreds μm and an outer diameter of a few hundreds μm. In order to improve the strength of the capillary102, the surface is covered with a polyimide coating. However, the polyimide coating is removed from the detecting unit113and the vicinity of the detecting unit113, to which laser light is applied. The inside of the capillary102is filled with a separation medium that separates DNA molecules in the sample. The separation medium is a polyacrylamide separation gel, for example.

The pump mechanism103is composed of a syringe105and a mechanical system that pressurizes the syringe105. The gel block106is a connecting unit that joins the syringe105, the capillary array114, an anode buffer container108, and a separation medium container107. The motor-operated valve110is closed, the syringe105is pressed in, and the separation medium in the syringe105is injected into the inside of the capillary102.

The thermostat115has a heater117and a fan116for controlling the temperature of the capillary array114, and is covered with a heat insulator ion order to keep the temperature in the thermostat115constant. Controlling the temperature in the thermostat115keeps the temperature in the most part of the capillary array114at a constant temperature, at a temperature of 60° C., for example.

The conveyor122has three electric motors and linear actuators, and the conveyor122is movable in three axial directions, vertical, lateral, and back-and-forth directions. On a moving stage123on the conveyor122, at least one more containers are installed. The conveyor122transports a buffer container118, a washing container119, a waste fluid container120, and a biochemical cartridge121on the moving stage123to the cathode end126of the capillary102. In the buffer container118, an electrophoretic buffer solution is put. The washing container119is used for washing the capillary102. Into the waste fluid container120, the separation medium in the capillary102is discharged. In the biochemical cartridge121, a biological sample, nucleic acid and a reagent, for example, is put. The nucleic acid amplified in the biochemical cartridge121is taken in from the cathode end126of the capillary102into the capillary array114. The biochemical cartridge121will be described later referring toFIG.2toFIG.5.

The high-voltage power supply104is connected to an anode electrode109in the anode buffer container108and the load header124, and applies a high voltage to the separation medium in the capillary102.

The light source111applies laser light that is coherent light as pumping light to the detecting unit113. The optical detector112optically detects fluorescence emitted from the sample in the detecting unit113. The detected optical data128is transferred to the control computer125through a control substrate127.

Referring toFIG.2, the biochemical cartridge121will be described.FIG.2is a perspective view of the biochemical cartridge121. The biochemical cartridge121is provided with one or a plurality, four, for example, of passages in which nucleic acid is amplified, and the cathode end126of the capillary102is inserted into each of the passages. Note that inFIG.2, the long direction of the passage is an X-direction, the direction in which the passages are arranged is a Y-direction, and the direction into which the cathode end126is inserted is a Z-direction.

Referring toFIG.3, the structure in the biochemical cartridge121will be described.FIG.3is a plan view of the inside of the biochemical cartridge121. In the biochemical cartridge121inFIG.3, a sample chamber301, a reagent chamber302, a sample passage303, and a reagent passage304are provided. A plurality, four, for example, of the sample chambers301are provided, and one microliter, for example, of a sample containing a biological sample is put in the sample chambers301. Alternatively, ten microliters of a sample may be put in the sample chamber301, and one microliter of the sample may be separated from ten microliters of the same for use. There is one or a plurality of the reagent chambers302. For example, in the case in which nucleic acid is amplified, five reagent chambers302are provided. In the reagent chambers302, reagents used for amplifying the nucleic acid, are put, including a primer, dNTP, a buffer solution, water, an enzyme, a denaturing agent, and a size standard DNA, for example.

The sample passages303are individually connected to the sample chambers301, and a droplet containing nucleic acid is transported. In the present embodiment, the direction in which a droplet containing nucleic acid is transported is the X-direction. In the case in which a technique of Electro Wetting On Dielectric (EWOD) is used for transporting the droplet, the sample passage303is a passage having an EWOD electrode300for transporting the droplet. EWOD is a technique in which a voltage is applied across a droplet disposed on a water-repellent film that is a film of water repellency and an EWOD electrode that is an electrode provided under the water-repellent film, the surface tension of the droplet is controlled, and thus the droplet is transported.

Referring toFIG.4, and example passage using EWOD will be described.FIG.4is a X-Z cross sectional view of a passage using EWOD. The passage using EWOD has a top plate401, an upper electrode402, an upper water-repellent film403, a lower water-repellent film405, an insulating film406, an EWOD electrode300, and an under plate407. The top plate401and the under plate407are disposed in parallel. On the under surface of the top plate401, the upper electrode402and the upper water-repellent film403are provided, and on the top surface of the under plate407, a plurality of EWOD electrodes300, the insulating film406, and the lower water-repellent film405are provided. Note that when a plurality of EWOD electrodes300is disposed at least one of the top plate401and the under plate407, the transportation of a droplet400is possible.

The plurality of EWOD electrodes300is arranged along the direction in which the droplet400is transported. The EWOD electrode300is covered with the insulating film406having a thickness of a few hundreds μm, for example, such that a voltage can be individually applied to the EWOD electrodes300. Preferably, a space between the upper water-repellent film403and the lower water-repellent film405is filled with a fluid404that is not mixed with the droplet400to be transported. Note that the transportation of the droplet400is possible with no the fluid404filled.

In such a passage using EWOD, when a voltage of a few tens of volts is applied to the EWOD electrode300located near the droplet400, the surface tension of the droplet400on the side of the EWOD electrode300to which the voltage is applied is changed, and an internal pressure is generated in the droplet400. Since the generated internal pressure drives the droplet400in the direction of an arrow inFIG.4, the droplet400is transported. That is, the droplet400is transported to the side of the EWOD electrode300to which the voltage is applied.

Again referring to the description ofFIG.3. The reagent passages304are individually connected to the reagent chambers302, and the droplet of the reagent is transported. In the present embodiment, the direction in which the droplet of the reagent is transported is the Y-direction. In the case in which EWOD is used for transporting the droplet of the reagent, the reagent passage304has a plurality of EWOD electrodes300similarly to the sample passage303. The reagent passage304intersects with the sample passage303, and the droplet containing nucleic acid is mixed with the droplet of the reagent at the intersection of the reagent passage304and the sample passage303. Note that an angle at which the reagent passage304intersects with the sample passage303is not limited to an angle of 90 degrees as shown inFIG.3.

The EWOD electrodes300on the sample passage303and the reagent passage304can separately apply a voltage, and thus two or more droplets can also be transported simultaneously. The direction in which the droplet is transported is not limited to one direction, and the droplet may be reciprocated. For example, mixing nucleic acid with the reagent may be promoted by reciprocating the droplet between the intersection of the sample passage303and the reagent passage304and the point adjacent to the intersection.

In the midway point of the sample passage303, a temperature control region305is provided. The temperature control region305is one or more regions in which the temperature is kept at a predetermined temperature, a region kept at a temperature of 60° C., for example, and a region kept at a temperature of 95° C. The droplet having the nucleic acid and the reagent mixed is transported to the temperature control region305, and the nucleic acid is amplified by a Polymerase Chain Reaction (PCR) or a cycle sequence reaction, for example. Note that the droplet may be reciprocated between the regions kept at different temperatures, the region at a temperature of 60° C. and the region at a temperature of 95° C., for example. The droplet having the nucleic acid amplified is labeled to be a sample droplet.

At the tip of the sample passage303, a droplet retaining unit306is provided. The droplet retaining unit306has a plurality of sample injection points, at ten places, for example. Each of the sample injection points includes the EWOD electrode300to control a voltage applied to the EWOD electrode300, and thus the sample droplet is transported to the position of a desired sample injection point, and the sample droplet is retained at the position. Preferably, the spacing between the EWOD electrodes300of the droplet retaining unit306is the same as the spacing between the EWOD electrodes300of the sample passage303or the reagent passage304. Providing the same spacing facilitates the manufacture of the EWOD electrode300.

ReferringFIG.5, the droplet retaining unit306according to the present embodiment will be described.FIG.5is a X-Z cross sectional view of the droplet retaining unit306. The droplet retaining unit306has a septum500, the top plate401, the upper electrode402, the upper water-repellent film403, the lower water-repellent film405, the insulating film406, the EWOD electrode300, and the under plate407. The lower water-repellent film405, the insulating film406, the EWOD electrode300, and the under plate407are the same configurations shown inFIG.4, and the description is omitted.

The top plate401, the upper electrode402, and the upper water-repellent film403are the same configurations shown inFIG.4except having an opening501. The opening501is opened at the position at which each of the EWOD electrodes300is disposed on the droplet retaining unit306along the Z-direction. That is, the EWOD electrode300of the droplet retaining unit306and the opening501have the same numbers.

The septum500is a rubber member disposed so as to cover the top surface of the top plate401, and has a hole into which the cathode end126of the capillary102is inserted. The parts of the septum500having the hole are individually inserted into the openings501. That is, one sample injection point includes one EWOD electrode300of the droplet retaining unit306, the opening501opened above the EWOD electrode300, and the hole of the septum500inserted into the opening501.

When a sample droplet502is transported to the position of a desired sample injection point, the cathode end126of the capillary102is inserted at the position until the cathode end126contacts the sample droplet502.FIG.5exemplifies a state in which the sample droplet502to which number1is given is in contact with the cathode end126. In this state, a voltage of a few kilovolts is applied to the load header124or a short time, and nucleic acid in the sample droplet502is taken in the inside of the capillary array114. The nucleic acid taken in the inside of the capillary array114is guided to the detecting unit113, pumping light is applied, and fluorescence is detected.

At the sample injection point used for taking in the nucleic acid in the sample droplet502, the insulating film406or the EWOD electrode300is destroyed due to a few kilovolts of an application voltage, and become non-reusable. Therefore, in the present embodiment, a plurality of sample injection points is provided, the sample injection point used for taking in the nucleic acid is changed every time when the nucleic acid is taken in. That is, the droplet retaining unit306according to the present embodiment has a plurality of EWOD electrodes300for retaining the sample droplet502, and a different EWOD electrode300is used every time when the nucleic acid in the sample droplet502is taken in the inside of the capillary array114. According to the present embodiment, EWOD can be used a plurality of times for taking in the nucleic acid by the capillary array114.

Note that for taking in the nucleic acid by the capillary array114, preferably, the EWOD electrode300is used in order of the EWOD electrode300at the tail end, i.e., in order of the number given to the sample droplet502inFIG.5, for example. with the use of the plurality of EWOD electrodes300in such order, the EWOD electrodes300of the droplet retaining unit306can be fully used.

In the present embodiment, the case is described in which nucleic acid, DNA, is specifically handled as an example of a biological sample. However, biological samples handled in the present invention is not limited to this, including general biological materials such as RNA, protein, polysaccharides, and microorganisms. For taking in a biological sample, a component other than the capillary102may be used.

Second Embodiment

In the first embodiment, the description is made in which the spacing between the openings501into which the cathode end126of the capillary102is inserted is the same as the spacing between the EWOD electrodes300of the droplet retaining unit306. In the case in which the spacing between the EWOD electrodes300of the droplet retaining unit306is too narrow, the area around the EWOD electrode300used for taking in the nucleic acid by the capillary array114is sometimes destroyed due to the application voltage to the load header124. Therefore, in the present embodiment, a configuration will be described in which the spacing between openings501is widened, EWOD can be used for taking in the nucleic acid a plurality of times even in the case in which the area around an EWOD electrode300used for taking in the nucleic acid is destroyed.

Referring toFIG.6, a droplet retaining unit306according to the present embodiment will be described. Similarly toFIG.5,FIG.6is a X-Z cross sectional view of the droplet retaining unit306. Similarly to the first embodiment, the droplet retaining unit306has a septum500, a top plate401, an upper electrode402, an upper water-repellent film403, a lower water-repellent film405, an insulating film406, an EWOD electrode300, and an under plate407, and the upper water-repellent film403is provided with an opening501. However, the opening501according to the present embodiment is provided in a spacing wider than the EWOD electrode300, in a spacing with no influence of destruction due to the application voltage to a load header124, for example. The opening501is provided in such a spacing, and thus the EWOD electrode300under the opening501adjacent to the opening501having been used for taking in the nucleic acid is not destroyed even in the case in which the area around the EWOD electrode300used for taking in the nucleic acid in the sample droplet502is destroyed. As a result, the nucleic acid can be taken in the capillary array114through the opening501above the EWOD electrode300that has not been destroyed.

According to the present embodiment, even in the case in which the area around the EWOD electrode300used for taking in the nucleic acid in the sample droplet502is destroyed, the EWOD electrode300under the opening501is not destroyed, and thus EWOD can be for taking in the nucleic acid a plurality of times.

Third Embodiment

In the first embodiment, the description is made in which the top plate401, the upper electrode402, and the upper water-repellent film403have the openings501in the same number as the number of the sample injection points. In the present embodiment, a configuration will be described in which a top plate401, an upper electrode402, and an upper water-repellent film403have an opening701shared at all sample injection points.

Referring toFIG.7, a droplet retaining unit306according to the present embodiment will be described. Similarly toFIG.5,FIG.7is a X-Z cross sectional view of the droplet retaining unit306. In the present embodiment, the top plate401, the upper electrode402, and the upper water-repellent film403have one opening701shared in the droplet retaining unit306. A septum500is disposed so as to block the opening701. The septum500has an opening into which a cathode end126of a capillary102is inserted. The opening of the septum may be a plurality of holes702opposite to the individual EWOD electrode300, o may be a slit703(seeFIG.8) across a plurality of EWOD electrodes300. The shared opening701is provided, and thus the biochemical cartridge121and the septum500can be formed in a simpler configurations.

Note that the biochemical analysis device according to the present invention is not limited to the foregoing embodiments, and can be embodied with the components modified in the scope not deviating from the gist of the invention. A plurality of components disclosed in the foregoing embodiments may be appropriately combined. Some components may be removed from all the components shown in the foregoing embodiments.

REFERENCE SIGNS LIST

101: device main body102: capillary103: pump mechanism104: high-voltage power supply105: syringe106: gel block107: separation medium container108: anode buffer container109: anode electrode110: motor-operated valve111: light source112: optical detector113: detecting unit114: capillary array115: thermostat116: fan117: heater118: buffer container119: washing container120: waste fluid container121: biochemical cartridge122: conveyor123: moving stage124: load header125: control computer126: cathode end127: control substrate128: optical data129: capillary head300: EWOD electrode301: sample chamber302: reagent chamber303: sample passage304: reagent passage305: temperature control region306: droplet retaining unit400: droplet401: top plate402: upper electrode403: upper water-repellent film404: fluid405: lower water-repellent film406: insulating film407: under plate500: septum501: opening502: sample droplet701: opening702: hole703: slit