阅读说明：本技术 试样处理方法、试样处理装置、程序及试样处理盒 (Sample processing method, sample processing device, program, and sample processing cartridge ) 是由 三浦由宣 山下秀治 长谷川雄大 林智也 于 2019-08-30 设计创作，主要内容包括：本发明旨在在使用盒处理试样时,可更简便地制成乳液。此试样处理方法是使用具备用于收容液体的室11的盒100的试样处理方法,其通过使盒100的室11收容试样80及分散剂82,使盒100绕旋转轴221旋转,搅拌室11内的试样80及分散剂82,形成含试样80的分散质分散于分散剂82中的乳液83。(The present invention aims to make an emulsion more easily when a sample is processed using a cartridge. This sample processing method is a sample processing method using a cartridge 100 having a chamber 11 for storing a liquid, and is a sample processing method in which a sample 80 and a dispersant 82 are stored in the chamber 11 of the cartridge 100, the cartridge 100 is rotated around a rotation shaft 221, the sample 80 and the dispersant 82 in the chamber 11 are stirred, and an emulsion 83 in which a dispersoid containing the sample 80 is dispersed in the dispersant 82 is formed.)

1. A sample processing method using a cartridge provided with a chamber for storing a liquid, comprising:

the chamber of the cartridge contains a sample and a dispersant,

the sample and the dispersant in the chamber are stirred by rotating the cartridge about a rotation axis, thereby forming an emulsion in which a dispersoid containing the sample is dispersed in the dispersant.

2. The sample processing method according to claim 1, wherein the cartridge includes a plurality of the chambers arranged in a substantially equal arc shape with respect to a radial direction of the rotation axis.

3. The sample processing method according to claim 1 or 2, wherein the emulsion is formed in the chamber by repeating an operation of changing a rotation speed of the cartridge rotating about a rotation axis.

4. The sample processing method according to claim 3, wherein the operation of changing the rotation speed of the cartridge includes an operation of reversing the rotation direction of the cartridge or an operation of accelerating and decelerating the cartridge in the same direction.

5. The sample processing method according to claim 4, wherein the operation of changing the rotation speed of the cartridge includes an operation of reversing the rotation direction of the cartridge in a cycle of 165 milliseconds to 330 milliseconds.

6. A sample treatment method as claimed in any one of claims 3 to 5, wherein the ratio of the total volume of said dispersoid and said dispersant in said chamber is 30% to 70%.

7. The sample processing method according to claim 3, wherein the action of changing the rotation speed of the cartridge comprises an action of reversing the rotation direction of the cartridge at a cycle of 330 milliseconds,

the ratio of the total volume of the dispersoid and the dispersant in the chamber is 30%.

8. The sample processing method of claim 3, wherein

The action of changing the rotational speed of the cartridge includes an action of reversing the rotational direction of the cartridge at a cycle of 165 msec,

the ratio of the total volume of the dispersoid and the dispersant in the chamber is 50% to 70%.

9. The sample treatment method according to any one of claims 1 to 8, wherein the substance to be detected contained in the sample is a nucleic acid or a protein.

10. A method of treating a sample as claimed in any one of claims 1 to 9, wherein

Treating the substance to be detected with a reagent containing a labeling substance for labeling the substance to be detected contained in the sample,

detecting a signal based on the labeling substance.

11. A method of treating a sample as claimed in any one of claims 1 to 10, wherein

The chamber is configured to accommodate the sample, the dispersant, and a reagent for treating a substance to be detected in the sample,

by rotating the cartridge, an emulsion is formed in which the dispersoid containing the sample and the reagent is dispersed in the dispersant.

12. The sample processing method of claim 11, wherein the reagent comprises:

a nucleic acid amplification reagent for amplifying the nucleic acid in the sample, or

A substrate that reacts with a labeling substance that specifically binds to a protein in the sample.

13. The sample treatment method according to claim 11 or 12, wherein the dispersant contains an oil that is immiscible with the sample and the reagent.

14. The sample processing method according to any one of claims 11 to 13, wherein the emulsion containing droplets of the dispersoid containing 1 molecule or 1 substance to be detected is formed in the chamber by rotation of the cartridge.

15. A method of sample treatment as claimed in any one of claims 11 to 14 wherein

The cartridge is connected to the chamber and includes a liquid storage portion for storing the liquid transferred to the chamber,

the liquid containing part comprises a No. 1 liquid containing part for containing the dispersing agent,

the dispersant is transferred from the 1 st liquid storage unit to the chamber by rotating the cartridge about a rotation axis.

16. The sample processing method according to any one of claims 11 to 15, wherein the substance to be detected and the reagent contained in the dispersoid are reacted by changing the temperature of the cartridge after the emulsion is formed.

17. The sample processing method of claim 12, wherein

The substance to be detected is a nucleic acid,

the reagent is a nucleic acid amplification reagent for amplifying a nucleic acid in the sample,

after the emulsion is formed, the nucleic acid contained in the dispersoid is amplified by periodically changing the temperature of the cartridge over a plurality of temperature ranges.

18. The sample processing method of claim 17, wherein

The cartridge is connected to the chamber and includes a liquid storage portion for storing the liquid transferred to the chamber,

the liquid containing part is connected with the chamber and contains a No. 2 liquid containing part for containing a reagent for demulsifying the emulsion by mixing,

after amplifying the nucleic acid contained in the dispersoid, a reagent for demulsifying the emulsion is transferred from the 2 nd liquid reservoir to the chamber in which the emulsion is formed by rotating the cartridge about a rotary shaft.

19. The sample processing method of claim 18, wherein

The liquid containing portion includes a closing body for closing the liquid containing portion,

after amplifying the nucleic acid contained in the dispersoid, unsealing the closure of the 2 nd liquid holding portion.

20. The sample processing method of claim 18 or 19, wherein the cartridge comprises:

the chamber of the emulsion is formed,

a 2 nd chamber connected to the chamber, and

a 3 rd liquid storage part connected to the 2 nd chamber for storing a reagent for washing the substance to be detected,

after amplifying at least the nucleic acid contained in the dispersoid, the reagent for washing the substance to be detected is transferred from the 3 rd liquid storage part to the 2 nd chamber by rotating the cartridge about a rotation axis.

21. The sample processing method of claim 20, wherein

The reagent for washing the detected substance contains alcohol,

the temperature of the cassette is changed within a range of 30 ℃ to 90 ℃ when the nucleic acid contained in the dispersoid is amplified.

22. The sample processing method of claim 20 or 21, wherein

The reagent contains magnetic particles that bind to nucleic acids in the sample,

after transferring a reagent for demulsifying the emulsion to the chamber, the nucleic acid bound to the magnetic particles is transferred from the chamber to the 2 nd chamber by applying a magnetic force to the cartridge.

23. The sample processing method of any of claims 20 to 22, wherein the cartridge comprises:

a 3 rd chamber connected to the 2 nd chamber, and

a 4 th liquid reservoir part connected to the 3 rd chamber for containing a reagent for denaturing the nucleic acid,

after amplifying at least the nucleic acid contained in the dispersoid, the reagent for denaturing the nucleic acid is transferred from the 4 th liquid reservoir unit to the 3 rd chamber by rotating the cartridge about a rotation axis.

24. The sample processing method of claim 23, wherein the cartridge comprises:

a 4 th chamber connected to the 3 rd chamber, and

a 5 th liquid reservoir part connected to the 4 th chamber for containing a reagent containing a labeling substance that reacts with the amplification product of the nucleic acid,

transferring a reagent containing the labeling substance from the 5 th liquid storage portion to the 4 th chamber by rotating the cartridge about a rotation axis,

the nucleic acid transferred to the 4 th chamber and the labeling substance are reacted by changing the temperature of the cassette.

25. The sample processing method of claim 24, wherein

Amplifying the nucleic acid contained in the dispersoid in the chamber by periodically changing the temperature of the cartridge at least in a plurality of temperature ranges by heating,

reacting the nucleic acid and the labeling substance in the 4 th chamber by raising the temperature of the cartridge at least by heating.

26. The sample processing method according to any one of claims 17 to 25, wherein the temperature of the cartridge is changed by bringing at least a temperature adjusting portion, which changes the temperature of the cartridge by heating, into contact with the cartridge.

27. A sample processing device is provided with:

a setting part for setting a cartridge having a chamber for accommodating the sample and the dispersant,

a rotation mechanism for rotating the cartridge set in the setting section around a rotation axis, an

And a control unit configured to stir the sample and the dispersant in the chamber by rotating the cartridge containing the sample and the dispersant in the chamber by the rotation mechanism, thereby controlling formation of an emulsion in which a dispersoid containing the sample is dispersed in the dispersant.

28. The sample processing apparatus of claim 27, wherein the control portion controls the rotation mechanism to repeat an operation of changing a rotation speed of the cartridge rotating about the rotation axis, thereby forming the emulsion in the chamber.

29. The sample processing apparatus according to claim 28, wherein the control unit controls the rotation mechanism so as to repeat an operation of reversing a rotation direction of the cartridge or an operation of accelerating and decelerating the cartridge in the same direction when the emulsion is formed.

30. The sample processing apparatus according to claim 29, wherein the controller controls the rotating mechanism so as to repeat an operation of reversing the rotation direction of the cartridge at a cycle of 165 milliseconds to 330 milliseconds when the emulsion is formed.

31. The sample processing device according to any one of claims 27 to 30, wherein the control unit forms the emulsion containing droplets of the dispersoid containing 1 molecule or 1 detected substance in the chamber by rotation of the cartridge.

32. The sample processing apparatus according to any one of claims 27 to 31, wherein the controller controls the rotating mechanism so as to transfer the dispersant contained in the 1 st liquid containing unit provided in the cartridge to the chamber by rotation.

33. The sample processing apparatus as claimed in any one of claims 27 to 32, wherein the control unit forms the emulsion in which the dispersoid containing the sample and the reagent is dispersed in the dispersing agent by rotating the cartridge containing the sample, the reagent for processing the substance to be detected in the sample, and the dispersing agent in the chamber by the rotating mechanism.

34. The sample processing apparatus according to claim 33, further comprising a temperature adjustment unit that changes the temperature of the cartridge to cause the substance to be detected contained in the dispersoid in the emulsion to react with the reagent.

35. The sample processing device of claim 34, wherein

The substance to be detected is a nucleic acid,

the reagent is a nucleic acid amplification reagent for amplifying a nucleic acid in the sample,

the temperature adjustment unit periodically changes the temperature of the cassette in a plurality of temperature ranges, thereby amplifying the nucleic acid contained in the dispersoid.

36. The sample processing apparatus according to claim 35, wherein the controller controls the rotating mechanism so as to transfer a reagent for demulsifying the emulsion from a 2 nd liquid reservoir provided in the cartridge to the chamber in which the emulsion is formed by rotation.

37. The sample processing device of claim 36, wherein

An unsealing mechanism is further provided for unsealing a sealing body for sealing the liquid storage portion of the cartridge,

the control unit controls the unsealing mechanism to form the emulsion in the chamber, change the temperature of the cartridge, react the substance to be detected and the reagent, and then unseal the sealed body.

38. The sample processing apparatus according to claim 37, further comprising a transfer mechanism for transferring the substance to be detected to a 2 nd chamber connected to the chamber,

the control unit performs the following control:

the rotation mechanism is controlled to transfer a reagent for washing the substance to be detected from a 3 rd liquid storage unit provided in the cartridge to the 2 nd chamber by rotation,

the transfer mechanism is controlled so that the substance to be detected is transferred from the chamber to the 2 nd chamber after the emulsion is demulsified.

39. The sample processing apparatus according to claim 38, wherein the control unit controls the unsealing mechanism to unseal the enclosure of the liquid holding portion that holds a reagent for washing the substance to be detected after at least the substance to be detected and the reagent are reacted by the temperature adjustment unit.

40. The sample processing device of claim 38 or 39, wherein

The reagent for washing the detected substance contains alcohol,

the control unit controls the temperature adjustment unit so that the temperature of the cartridge is changed within a range of 30 ℃ to 90 ℃.

41. The sample processing apparatus according to any one of claims 38 to 40, wherein the control unit performs control such that:

controlling the rotation mechanism so as to transfer a reagent for denaturing nucleic acid from a 4 th liquid storage unit provided in the cartridge to a 3 rd chamber connected to the 2 nd chamber by rotation,

the transfer mechanism is controlled to transfer the substance to be detected stored in the 2 nd chamber to the 3 rd chamber.

42. The sample processing apparatus according to claim 41, wherein the control unit controls the unsealing mechanism so as to unseal the enclosure of the liquid holding portion that holds the reagent for denaturing the nucleic acid after at least the substance to be detected and the reagent are reacted by the temperature adjustment unit.

43. The sample processing apparatus according to claim 41 or 42, wherein the control section performs control such that:

controlling the rotation mechanism so as to transfer a reagent containing a labeling substance that reacts with the amplification product of the nucleic acid from a 5 th liquid storage unit provided in the cassette to a 4 th chamber connected to the 3 rd chamber by rotation,

the transfer mechanism is controlled to transfer the substance to be detected accommodated in the 3 rd chamber to the 4 th chamber.

44. The sample processing apparatus of claim 43, wherein the temperature adjustment section performs:

amplifying the nucleic acid contained in the dispersoid in the chamber by periodically changing the temperature of the cartridge over a plurality of temperature ranges,

reacting the nucleic acid and the labeling substance in the 4 th chamber by raising the temperature of the cartridge.

45. The sample processing apparatus according to any one of claims 34 to 44, wherein the temperature adjustment unit is configured to be relatively movable to a position away from a position in contact with the cartridge disposed in the setting unit, and changes the temperature of the cartridge in a state of contact with the cartridge.

46. The sample processing device of claim 34, wherein

The substance to be detected is a protein,

the reagent contains a substrate that reacts with a labeling substance that specifically binds to a protein in the sample.

47. A recording medium on which a program for causing a computer to function as a control unit for performing control is recorded,

the computer is connected with the sample processing device,

the sample processing apparatus processes a sample using a cartridge having a chamber for containing a liquid,

the control is as follows:

the cartridge in which the sample and the dispersant are accommodated in the chamber is rotated around a rotation axis by a rotation mechanism provided in the sample processing apparatus,

the sample and the dispersant in the chamber are stirred by rotating the cartridge, thereby forming an emulsion in which a dispersoid containing the sample is dispersed in the dispersant.

48. A sample processing cartridge comprising:

a sample introduction portion for introducing a sample, an

A chamber for accommodating the introduced sample and the dispersing agent,

the sample processing cartridge is configured to stir the sample and the dispersant in the chamber by rotating around a rotation axis to form an emulsion in which a dispersoid containing the sample is dispersed in the dispersant.

49. The sample processing cartridge of claim 48, further comprising a liquid storage portion which is fluidly connected to the chamber and in which the dispersant is stored in advance,

the sample processing cartridge is configured to transfer the dispersant from the liquid storage portion to the chamber by rotating about the rotation axis.

[ technical field ] A method for producing a semiconductor device

The present invention relates to a sample processing method for processing a sample using a cartridge having a housing space for housing a liquid (see, for example, patent document 1).

[ background of the invention ]

Patent document 1 discloses a technique of forming an emulsion containing a biological sample by transferring a liquid into a cartridge 900 having a plurality of holes 901 for holding the liquid, as shown in fig. 41. In fig. 41, the cartridge 900 includes 4 wells 901, i.e., 2 oil wells 901a, 1 sample well 901b, and 1 collection well 901 c. The wells 901 are connected via a fluidic channel 902 formed in the cartridge 900. Each hole 901 is open at the upper part. Oil is supplied from 2 oil wells 901a, and a mixture of a sample and a reagent is supplied from a sample well 901 b. The oil or the mixed liquid contained in the hole 901 is injected from an opening in the upper part of the hole 901, and the liquid in the hole 901 is transferred to the fluid channel 902 by applying air pressure from the opening. The liquid mixture and the oil join in the fluid passage 902, an emulsion in which droplets of the liquid mixture are dispersed in the oil is formed, and the formed emulsion is stored in the recovery hole 901 c.

[ Prior art documents ]

[ patent document ]

[ patent document 1 ] specification of U.S. Pat. No. 9126160

[ SUMMARY OF THE INVENTION ]

[ problem to be solved by the invention ]

In the method of forming an emulsion by feeding a mixed liquid or oil to a fluid channel by applying air pressure to a hole as in the above-mentioned patent document 1, each droplet is formed in the mixed liquid by shearing the flow of the mixed liquid from the flow of the oil at the confluence portion of the fluid channel. Therefore, for 1 well, it becomes troublesome to finely adjust the air pressure in accordance with the viscosity of each liquid flowing through the fluid channel or the affinity of the liquids with each other. In addition, in the case of configuring a sample processing apparatus for executing such a sample processing method, it is necessary to provide a mechanism for adding air pressure to 1 well or a mechanism for precisely controlling the air pressure, and the apparatus configuration becomes complicated. It is therefore desirable that when processing a sample using a cartridge, it becomes possible to make an emulsion more easily.

The object of the present invention is to make it easier to prepare an emulsion when a cartridge is used to process a sample.

[ MEANS FOR SOLVING PROBLEMS ] to solve the problems

According to the sample processing method of the invention 1, a sample processing method using a cartridge 100 having a chamber 11 for storing a liquid is provided, in which a sample 80 and a dispersant 82 are stored in the chamber 11 of the cartridge 100, the cartridge 100 is rotated around a rotation shaft 221, and the sample 80 and the dispersant 82 in the chamber 11 are stirred to form an emulsion 83 in which a dispersoid containing the sample 80 is dispersed in the dispersant 82.

The emulsion is a dispersion solution in which a dispersoid is dispersed in a dispersant. The dispersion system refers to a state in which the dispersoid is suspended or suspended in the dispersant. The dispersant and the dispersoid are both liquids. However, the dispersant and the dispersoid may be a solution other than a liquid. The dispersoid is not mixed with the dispersant. That is, the dispersant and dispersoid do not form a homogeneous phase upon mixing. The dispersoids are separated from each other by the dispersant and surrounded by the dispersant. Thus, droplets of dispersoids are formed in the dispersant in the emulsion. The formation of an emulsion is referred to as "emulsification". The state in which the emulsion is released to separate the dispersoid and the dispersant is referred to as "demulsification".

In the sample processing method according to the first aspect, the sample (80) and the dispersant (82) in the chamber (11) are stirred by rotating the cartridge (100) about the rotation axis (221) and applying an inertial force accompanying the rotation of the cartridge (100) to the chamber (11). The dispersoid is finely sheared by the dispersant (82) with stirring. As a result, an emulsion (83) is formed in which droplets (84) containing a dispersoid of the substance (MD) to be detected contained in the sample (80) are dispersed in the dispersing agent (82). Thus, since the emulsion (83) can be formed in the chamber (11) by only rotating the cartridge (100) about the rotation shaft (221) without using liquid feeding controlled by air pressure, the emulsion (83) can be more easily prepared when a sample is processed using the cartridge.

In the sample processing method according to the 1 st aspect, the cartridge (100) preferably includes a plurality of chambers (11) arranged in a circular arc shape having substantially the same distance from the radial direction of the rotation axis (221). With this configuration, emulsions (83) containing the same or different samples can be prepared in each of the plurality of chambers (11) only by rotating the cartridge (100) about the rotation shaft (221). In this case, when the distances from the rotating shafts (221) of the respective chambers (11) are different, the magnitudes of the inertial forces acting on the respective chambers (11) are different, and the quality of the produced emulsion (83) may be disturbed for each chamber (11). In contrast, in the above configuration in which the plurality of chambers (11) are arranged in an arc shape in which the distances from the radial direction of the rotation axis (221) are substantially equal, the magnitudes of the inertial forces acting on the respective chambers (11) along with the rotation become substantially equal, and the quality of the emulsion (83) to be produced can be made more uniform. The quality of the emulsion (83) refers to, for example, the number or size (or diameter) of droplets of the dispersoid contained in the emulsion (83).

In the sample processing method according to the 1 st aspect, it is preferable that the emulsion (83) is formed in the chamber (11) by repeating an operation of changing the rotation speed of the cartridge (100) rotating around the rotation shaft (221). With this configuration, by repeating the operation of changing the rotational speed of the cartridge (100), the inertial force accompanying the speed change acts to efficiently stir the sample (80) and the dispersant (82) in the chamber (11), and the dispersoid can be rapidly and finely sheared by the dispersant (82). As a result, the emulsion (83) can be efficiently produced.

In the configuration in which the operation of changing the rotational speed of the cartridge (100) is repeated, the operation of changing the rotational speed of the cartridge (100) preferably includes an operation of reversing the rotational direction of the cartridge (100) or an operation of accelerating and decelerating in the same direction. The operation of reversing the rotation direction of the cartridge includes rotation in one direction of the cartridge, stop of the rotation, and rotation in the other direction of the cartridge. The action of accelerating and decelerating in the same direction may include stopping the rotation by deceleration. With this configuration, when the rotation direction of the cartridge (100) is reversed, a large inertial force can be applied when the rotation direction is reversed, and the emulsion (83) can be efficiently produced. Since it is not necessary to reversely rotate a rotation mechanism for rotating the cartridge (100) when the cartridge (100) is accelerated and decelerated in the same direction, the emulsion (83) can be easily produced.

In this case, the operation of changing the rotational speed of the cartridge (100) preferably includes an operation of reversing the rotational direction of the cartridge (100) in a cycle of 165 milliseconds to 330 milliseconds. As a result of experiments described later, the inventors of the present invention found that it is preferable to prepare an emulsion (83) for detection by processing a substance (MD) to be detected, by setting a cycle of reversing the rotation direction of a cartridge (100) to 165 milliseconds to 330 milliseconds. Therefore, the detection target substance (MD) can be processed by repeating the operation of inverting the rotation direction at a cycle of 165 milliseconds to 330 milliseconds, thereby producing an emulsion (83) suitable for detection.

In the configuration in which the operation of changing the rotational speed of the cartridge (100) is repeated, the ratio of the total volume of the dispersoid and the dispersant (82) in the chamber (11) is preferably 30% to 70%. Among them, the present inventors found, as a result of experiments described later, that it is preferable to prepare an emulsion (83) for detection by treating a substance (MD) to be detected by storing a liquid in a ratio of 30% to 70% in a chamber (11). Therefore, an emulsion (83) suitable for handling and detecting a substance (MD) to be detected can be prepared from the above-described constitution.

In the above configuration in which the operation of changing the rotational speed of the cartridge (100) is repeated, the operation of changing the rotational speed of the cartridge (100) preferably includes an operation of reversing the rotational direction of the cartridge (100) at a cycle of 330 milliseconds, and the ratio of the total volume of the dispersoids and the dispersant (82) in the chamber (11) is 30%. With this configuration, the substance (MD) to be detected can be further processed based on the test results described later to prepare an emulsion (83) suitable for detection.

In the configuration in which the operation of changing the rotational speed of the cartridge (100) is repeated, the operation of changing the rotational speed of the cartridge (100) preferably includes an operation of reversing the rotational direction of the cartridge (100) at a cycle of 165 milliseconds, and the ratio of the total volume of the dispersoids and the dispersant (82) in the chamber (11) is preferably 50% to 70%. With this configuration, the substance (MD) to be detected can be further processed based on the test results described later to prepare an emulsion (83) suitable for detection.

In the sample processing method according to the 1 st aspect, the substance (MD) to be detected contained in the sample (80) is preferably a nucleic acid or a protein. With this configuration, an emulsion (83) can be formed in which minute droplets (84) containing a dispersoid of a nucleic acid or a protein as a substance (MD) to be detected are dispersed in a dispersing agent (82). As a result, each protein or nucleic acid can be reliably processed or detected by partitioning each detection target substance (MD) within the droplet (84).

In the sample processing method according to the 1 st aspect, the substance (MD) to be detected may be treated with a reagent (88) containing a Labeling Substance (LS) for labeling the substance (MD) to be detected contained in the sample (80), and a signal based on the Labeling Substance (LS) may be detected.

In the sample processing method according to the 1 st aspect, it is preferable that the sample (80), the dispersing agent (82), and the reagent (81) for processing the substance (MD) to be detected in the sample (80) are stored in the chamber (11), and the cartridge (100) is rotated to form the emulsion (83) in which the dispersoid containing the sample (80) and the reagent (81) is dispersed in the dispersing agent (82). With this configuration, the substance (MD) to be detected can be treated with the reagent (81) in the droplets (84) of the dispersoid contained in the emulsion (83).

In this case, the reagent (81) preferably contains a nucleic acid amplification reagent for amplifying the nucleic acid in the sample (80) or a substrate that reacts with a labeling substance that specifically binds to the protein in the sample (80). With this configuration, amplification of nucleic acid as the test substance (MD) in the droplets (84) contained in the emulsion (83) or detection of a labeling substance based on protein as the test substance (MD) can be reliably performed for each test substance (MD).

In the sample treatment method according to claim 1, the dispersant (82) may be an oil that is immiscible with the sample (80) and the reagent (81).

In the formation of the emulsion (83) containing the dispersoid of the sample (80) and the reagent (81), it is preferable that the emulsion (83) containing the droplets (84) containing 1 molecule or 1 molecule of the dispersoid of the substance (MD) to be detected is formed in the chamber (11) by the rotation of the cartridge (100). With this configuration, each of 1 test substance (MD) contained in the sample can be separated and surely handled in the droplet (84) contained in the emulsion (83). As a result, even if the sample contains only a very small amount of the substance (MD) to be detected, the sample can be processed with high accuracy, and the detection accuracy can be improved when the processed substance (MD) to be detected is detected.

In the configuration of forming the emulsion (83) containing the dispersoid of the sample (80) and the reagent (81), the cartridge (100) preferably includes a liquid storage section (30) connected to the chamber (11) and storing the liquid transferred to the chamber (11), the liquid storage section (30) includes a 1 st liquid storage section (30) storing the dispersant (82), and the dispersant (82) is transferred from the 1 st liquid storage section (31) to the chamber (11) by rotating the cartridge (100) about a rotation shaft (221). With this configuration, not only the emulsion (83) is generated, but also the operation of storing the dispersant (82) in the chamber (11) can be performed by simply rotating the cartridge (100). Therefore, for example, since it is not necessary to add air pressure to the 1 st liquid storage part (31), the sample can be processed more easily.

In the constitution of forming the emulsion (83) containing the dispersoid of the sample (80) and the reagent (81), it is preferable that the substance (MD) to be detected contained in the dispersoid and the reagent (81) are reacted by changing the temperature of the cartridge (100) after the emulsion (83) is formed. With this configuration, the substance (MD) to be detected and the reagent (81) for processing the substance (MD) to be detected can be reliably reacted by a temperature change in a state where the substance (MD) to be detected and the reagent (81) to be detected are accommodated in the droplets (84) of the dispersoid and partitioned. In addition, in the liquid droplets (84) contained in the emulsion (83), the substance (MD) to be detected and the reagent (81) can be efficiently reacted by using a temperature change, compared with the case where the reaction of the substance (MD) to be detected and the reagent (81) is naturally performed at the temperature of the installation environment of the cartridge (100).

In the constitution of forming an emulsion (83) containing a dispersoid of a sample (80) and a reagent (81), the nucleic acid of the substance (MD) to be detected, and the reagent (81) is preferably a nucleic acid amplification reagent for amplifying the nucleic acid in the sample (80), and after the emulsion (83) is formed, the nucleic acid contained in the dispersoid is amplified by periodically changing the temperature of the cartridge (100) over a plurality of temperature ranges. With this configuration, the droplets (84) contained in the emulsion (83) can be subjected to a so-called thermal cycle treatment to efficiently amplify the nucleic acid.

In this case, the cartridge (100) preferably includes a liquid storage section (30) connected to the chamber (11) and storing the liquid transferred to the chamber (11), the liquid storage section (30) includes a 2 nd liquid storage section (32) connected to the chamber (11) and storing a reagent (85) for demulsifying the emulsion by mixing, and the reagent (85) for demulsifying the emulsion is transferred from the 2 nd liquid storage section (32) to the chamber (11) in which the emulsion (83) is formed by rotating the cartridge (100) around the rotation shaft (221) after the nucleic acid contained in the dispersoid is amplified. With this configuration, the nucleic acid amplified in the droplet 84 contained in the emulsion 83 can be taken out by the reagent 85 for demulsifying the emulsion. In this case, unlike the case of delivery by air pressure or the like, the reagent (85) for demulsifying the emulsion can be delivered to the chamber (11) only by rotating the cartridge (100) about the rotating shaft (221), and the emulsion (83) can be easily demulsified.

In the above configuration in which the cartridge (100) includes the liquid storage part (30), the liquid storage part (30) preferably includes a closure body (30a) that closes the liquid storage part (30), and after the nucleic acid contained in the dispersion is amplified, the closure body (30a) of the 2 nd liquid storage part (32) is opened. With this configuration, the sealing member (30a) prevents an agent (85) for demulsifying the emulsion from being unintentionally delivered from the 2 nd liquid reservoir unit (32). Therefore, it is possible to surely form the emulsion (83) and amplify the nucleic acid, and then demulsify the emulsion (83).

In the above-mentioned configuration in which the cartridge (100) includes the liquid storing part (30), the cartridge (100) preferably has a chamber (11) in which an emulsion (83) is formed, and a 3 rd liquid storing part (33) which includes a 2 nd chamber (12) connected to the chamber (11) and a reagent (86) for washing the substance (MD) to be detected and which is connected to the 2 nd chamber (12), and the reagent (86) for washing the substance (MD) to be detected is transferred from the 3 rd liquid storing part (33) to the 2 nd chamber (12) by rotating the cartridge (100) around a rotating shaft (221) after at least nucleic acid contained in the dispersoid is amplified. With this configuration, nucleic acid as a substance to be detected (MD) can be amplified in the chamber (11), and after nucleic acid is taken out from the droplet (84) by demulsification, the nucleic acid is washed in the 2 nd chamber (12). In addition, since the temperature change due to thermal cycling is accompanied in the process of amplifying nucleic acid in the chamber (11), in the state where the reagent (86) for washing the substance (MD) to be detected is contained in the 2 nd chamber (12), the reagent (86) for washing the substance (MD) to be detected may be affected by the temperature change. Thus, by transferring the reagent (86) for washing the substance (MD) to the 2 nd chamber (12) after amplification, the influence of temperature change on the reagent (86) for washing the substance (MD) can be suppressed.

In this case, the reagent (86) for washing the substance (MD) to be detected is preferably an alcohol, and the temperature of the cassette (100) is changed in the range of 30 ℃ to 90 ℃ when amplifying the nucleic acid contained in the dispersoid. Thus, when the temperature change during nucleic acid amplification is 30 ℃ to 90 ℃, the reagent (86) for washing the test substance (MD) may be vaporized by the influence of the temperature change. Therefore, the constitution of conveying the reagent (86) for washing the substance (MD) to be detected to the 2 nd chamber (12) after the nucleic acid amplification is particularly effective at a point where the vaporization of the alcohol can be suppressed.

In the above configuration in which the cartridge (100) includes a chamber (11) in which an emulsion (83) is formed and a 2 nd chamber (12) connected to the chamber (11), the reagent (81) preferably includes magnetic Particles (PM) that bind to nucleic acids in the sample (80), and after the reagent (85) that demulsifies the emulsion is transferred to the chamber (11), the nucleic acids that bind to the magnetic Particles (PM) from the demulsified liquid are transferred from the chamber (11) to the 2 nd chamber (12) by applying a magnetic force to the cartridge (100). With this configuration, the nucleic acid as the substance to be detected (MD) is bound to the magnetic Particles (PM), and the substance to be detected (MD) can be transferred to the 2 nd chamber (12) simply by applying a magnetic force to the cartridge (100) from the outside without being transported by air pressure or the like. Further, the substance (MD) to be detected in the liquid transfer chamber (11) is transferred by air pressure or the like, but the substance (MD) to be detected, which is bonded only to the magnetic Particles (PM), can be transferred to the 2 nd chamber (12) while leaving the liquid by transfer by magnetic force. As a result, the transfer of unnecessary components to the 2 nd chamber (12) is suppressed, and the nucleic acid in the 2 nd chamber (12) can be efficiently washed.

In the above-mentioned cartridge (100) comprising a chamber (11) in which an emulsion (83) is formed and a 2 nd chamber (12) connected to the chamber (11), the cartridge (100) preferably comprises a 3 rd chamber (13) connected to the 2 nd chamber (12) and a 4 th liquid reservoir part (34) connected to the 3 rd chamber (13) and containing a reagent (87) for denaturing a nucleic acid, and the reagent (87) for denaturing a nucleic acid is transferred from the 4 th liquid reservoir part (34) to the 3 rd chamber (13) by rotating the cartridge (100) around a rotation shaft (221) after at least a nucleic acid contained in a dispersoid is amplified. With this configuration, in the cartridge (100), a treatment for denaturing nucleic acid can be performed on the nucleic acid after the treatment for washing the nucleic acid in the 2 nd chamber (12). Further, even when the treatment for denaturing nucleic acid is performed, since the cassette (100) can be transported to the 3 rd chamber (13) only by rotating the cassette around the rotation shaft (221) without using a liquid transport such as air pressure, the treatment for denaturing nucleic acid can be performed easily.

In this case, the cartridge (100) preferably includes a 4 th chamber (14) connected to the 3 rd chamber (13) and a 5 th liquid containing portion (35) connected to the 4 th chamber (14) and containing a reagent (88) containing a Labeling Substance (LS) that reacts with an amplification product of a nucleic acid, and the nucleic acid transferred to the 4 th chamber (14) and the Labeling Substance (LS) are reacted by rotating the cartridge (100) about a rotating shaft (221) to transfer the reagent (88) containing the Labeling Substance (LS) from the 5 th liquid containing portion (35) to the 4 th chamber (14) and changing the temperature of the cartridge (100). In such a configuration, the cartridge (100) may be subjected to not only a process of denaturing nucleic acids but also a process of labeling for detecting nucleic acids as a detection target substance (MD). Further, in the process of labeling nucleic acids, it is possible to transfer the nucleic acids to the 4 th chamber (14) by simply rotating the cartridge (100) around the rotation shaft (221) without using a liquid transfer medium such as air pressure, and thus the process of labeling nucleic acids can be easily performed.

In the configuration in which the nucleic acid transferred to the 4 th chamber (14) is reacted with the Labeling Substance (LS) by changing the temperature of the cassette (100), it is preferable that the nucleic acid contained in the dispersoid in the chamber (11) is amplified by periodically changing the temperature of the cassette (100) at least in a plurality of temperature ranges by heating, and the nucleic acid in the 4 th chamber (14) is reacted with the Labeling Substance (LS) by increasing the temperature of the cassette (100) at least by heating. With this configuration, at least the cartridge (100) is heated to perform the process of amplifying the nucleic acid and the process of labeling the nucleic acid, and the process can be performed in the cartridge (100). Therefore, not only the preparation of the emulsion (83), but also the treatment of amplifying the nucleic acid and the treatment of labeling the nucleic acid using the cassette (100) can be performed more easily.

In the configuration in which the nucleic acid contained in the dispersoid in the chamber (11) is amplified by changing the temperature of the cartridge (100), it is preferable that the temperature of the cartridge (100) is changed by bringing at least a temperature adjusting portion (260) for changing the temperature of the cartridge (100) by heating into contact with the cartridge (100). With this configuration, the temperature of the cartridge (100) can be easily changed by efficiently transferring heat by bringing the temperature adjusting unit (260) into contact with the cartridge (100) as compared with the case of heating the cartridge (100) without contact.

The sample processing apparatus according to claim 2 of the present invention includes a setting unit (210) for setting a cartridge (100) having a chamber (11) for housing a sample (80) and a dispersant (82), a rotating mechanism (220) for rotating the cartridge (100) disposed in the setting unit (210) around a rotating shaft (221), and a control unit (230) for controlling the mixing of the sample (80) and the dispersant (82) in the chamber (11) and the formation of an emulsion (83) in which a dispersoid including the sample (80) is dispersed in the dispersant (82) by rotating the cartridge (100) in which the sample (80) and the dispersant (82) are housed in the chamber (11) by the rotating mechanism (220).

In the sample processing apparatus according to claim 2, the cartridge (100) is rotated about the rotation shaft (221), and the inertial force accompanying the rotation of the cartridge (100) acts on the chamber (11) to stir the sample (80) and the dispersant (82) in the chamber (11). The dispersoid is finely sheared by the dispersant (82) with stirring. As a result, an emulsion (83) is formed in which droplets (84) containing a dispersoid of the substance (MD) to be detected in the sample (80) are dispersed in the dispersing agent (82). Thus, an emulsion (83) can be formed in the chamber (11) by rotating the cartridge (100) only about the rotation shaft (221) without using liquid feeding controlled by air pressure, and the emulsion (83) can be more easily prepared when a sample is processed using the cartridge. Further, when the emulsion is formed by using the liquid feeding controlled by the air pressure, it is necessary to provide a mechanism for adding the air pressure to each of the portion for storing the dispersing agent (82) and the portion for storing the sample or a mechanism for precisely controlling the air pressure, and in the above configuration, the emulsion (83) can be formed only by providing the rotating mechanism (220), so that the apparatus configuration can be simplified, and a smaller and simpler sample processing apparatus can be obtained.

In the sample processing apparatus according to claim 2, the control unit (230) preferably controls the rotation mechanism (220) so as to repeat an operation of changing the rotation speed of the cartridge (100) rotating about the rotation axis (221), thereby forming the emulsion (83) in the chamber (11). With this configuration, by repeating the operation of changing the rotational speed of the cartridge (100), the inertial force accompanying the speed change acts to efficiently stir the sample (80) and the dispersant (82) in the chamber (11), and the dispersoid can be rapidly and finely sheared by the dispersant (82). As a result, the emulsion (83) can be efficiently produced.

In the configuration in which the operation of changing the rotational speed of the cartridge (100) is repeated, the control unit (230) preferably controls the rotating mechanism (220) so that the operation of reversing the rotational direction of the cartridge (100) or the operation of accelerating and decelerating in the same direction is repeated when the emulsion (83) is formed. With this configuration, when the rotation direction of the cartridge (100) is reversed, a large inertial force can be applied when the rotation direction is reversed, and thus the emulsion (83) can be efficiently produced. Since the rotation mechanism (220) does not need to be rotated reversely when the cartridge (100) is accelerated and decelerated in the same direction, the emulsion (83) can be easily prepared by simple control.

In this case, the control unit (230) preferably controls the rotation mechanism (220) so as to repeat an operation of reversing the rotation direction of the cartridge (100) in a cycle of 165 milliseconds to 330 milliseconds when the emulsion (83) is formed. With this configuration, based on the results of the experiment described later, the substance to be detected (MD) can be processed by repeating the operation of inverting the rotation direction in a cycle of 165 milliseconds to 330 milliseconds, thereby producing an emulsion (83) suitable for detection.

In the sample processing apparatus according to claim 2, the control unit (230) preferably forms an emulsion (83) containing droplets (84) of a dispersoid containing 1 molecule or 1 detection target substance (MD) in the chamber (11) by rotation of the cartridge (100). With this configuration, each of 1 test substance (MD) contained in the sample can be separated and surely handled in the droplet (84) contained in the emulsion (83). As a result, even if the sample contains only a very small amount of the substance (MD) to be detected, the sample can be processed with high accuracy, and the detection accuracy can be improved when the processed substance (MD) to be detected is detected.

In the sample processing apparatus according to claim 2, the control unit (230) preferably controls the rotation mechanism (220) so as to transfer the dispersant (82) stored in the 1 st liquid storage unit (31) provided in the cartridge (100) to the chamber (11) by rotation. In this configuration, not only the generation of the emulsion (83) but also the operation of storing the dispersant (82) in the chamber (11) can be performed only by rotating the cartridge (100) by the rotating mechanism (220). Therefore, for example, since it is not necessary to add air pressure to the 1 st liquid storage part (31), the sample can be processed more easily. Further, since the dispersant (82) is fed from the liquid storage section (30) to the chamber (11), a mechanism for adding air pressure is not required, and the device configuration can be further simplified.

In the sample processing apparatus according to claim 2, the control unit (230) preferably forms an emulsion (83) in which a dispersoid containing the sample (80) and the reagent (81) is dispersed in the dispersing agent (82) by rotating a cartridge (100) that contains the sample (80), the reagent (81) for processing the substance (MD) to be detected in the sample (80), and the dispersing agent (82) in the chamber (11) by a rotating mechanism (220). With this configuration, the substance (MD) to be detected can be treated with the reagent (81) in the droplets (84) of the dispersoid contained in the emulsion (83).

In this case, it is preferable that the cartridge (100) further includes a temperature adjusting section (260) for reacting the substance (MD) to be detected contained in the dispersoid in the emulsion (83) with the reagent (81) by changing the temperature of the cartridge. With this configuration, the substance (MD) to be detected and the reagent (81) for processing the substance (MD) to be detected can be reliably reacted by a temperature change in a state where the substance (MD) to be detected and the reagent (81) to be detected are accommodated in the droplets (84) of the dispersoid and partitioned. In addition, in the droplets (84) contained in the emulsion (83), the substance (MD) to be detected and the reagent (81) can be efficiently reacted by a temperature change, as compared with the case where the reaction of the substance (MD) to be detected and the reagent (81) is naturally performed at the temperature of the installation environment of the cartridge (100).

In the configuration provided with the temperature adjustment unit (260), it is preferable that the substance (MD) to be detected is a nucleic acid, the reagent (81) is a nucleic acid amplification reagent for amplifying the nucleic acid in the sample (80), and the temperature adjustment unit (260) amplifies the nucleic acid contained in the dispersoid by periodically changing the temperature of the cartridge (100) over a plurality of temperature ranges. With this configuration, the droplets (84) contained in the emulsion (83) can be subjected to a so-called thermal cycle treatment to efficiently amplify the nucleic acid.

In this case, the control unit (230) preferably controls the rotation mechanism (220) so as to transfer, by rotation, a reagent (85) for demulsifying an emulsion from a 2 nd liquid storage unit (32) provided in the cartridge (100) to the chamber (11) for forming the emulsion (83). In this configuration, not only the process of forming the emulsion (83) but also the process of demulsifying the emulsion (83) may be performed in the cartridge (100) by the sample processing apparatus (200). In this case, the device configuration can be further simplified because a mechanism for adding air pressure is not necessary for feeding the reagent (85) for demulsifying the emulsion into the chamber (11).

In the configuration in which the reagent (85) for demulsifying the emulsion is transferred by rotation from the 2 nd liquid storage portion (32) of the cartridge (100), it is preferable that an unsealing mechanism (250) is further provided for unsealing the sealing body (30a) that seals the liquid storage portion (30) of the cartridge (100), and the control unit (230) controls the unsealing mechanism (250) so that the emulsion (83) is formed in the chamber (11), the temperature of the cartridge (100) is changed to react the substance (MD) to be detected and the reagent (81), and then the sealing body (30a) is unsealed. With this configuration, the sealing member (30a) prevents an agent (85) for demulsifying the emulsion from being unintentionally delivered from the 2 nd liquid reservoir unit (32). Therefore, by providing an unsealing mechanism (250) for unsealing the closure (30a), it is possible to surely form the emulsion (83) and amplify the nucleic acid, and then to perform demulsification of the emulsion (83).

In this case, it is preferable that a transfer mechanism (240) for transferring the detection object (MD) to the 2 nd chamber (12) connected to the chamber (11) is further provided, and the control unit (230) controls the rotation mechanism (220) so as to transfer the reagent (86) for washing the detection object (MD) from the 3 rd liquid storage unit (33) provided in the cartridge (100) to the 2 nd chamber (12) by rotation, and controls the transfer mechanism (240) so as to transfer the detection object (MD) from the chamber (11) to the 2 nd chamber (12) after the emulsion (83) is demulsified. With this configuration, nucleic acid as a substance to be detected (MD) can be amplified in the chamber (11), and after nucleic acid is taken out from the droplet (84) by demulsification, the nucleic acid is washed in the 2 nd chamber (12).

In the configuration in which the reagent (86) for washing the substance (MD) to be detected is transferred from the 3 rd liquid storage portion (33) provided in the cartridge (100) to the 2 nd chamber (12) by rotation, the control unit (230) preferably controls the unsealing mechanism (250) so as to unseal the sealing body (30a) of the liquid storage portion (30) that stores the reagent (86) for washing the substance (MD) to be detected after the substance (MD) to be detected and the reagent (81) are reacted at least by the temperature adjustment unit (260). With this configuration, the reagent (86) for washing the test substance (MD) is transferred to the 2 nd chamber (12) after being amplified by a temperature change using the temperature adjustment unit (260), thereby suppressing the influence of the temperature change on the reagent (86) for washing the test substance (MD).

In this case, the reagent (86) for washing the test substance (MD) preferably contains an alcohol, and the control unit (230) controls the temperature adjustment unit (260) so that the temperature of the cartridge (100) is changed within a range of 30 ℃ to 90 ℃. Thus, when the temperature change during nucleic acid amplification is 30 ℃ to 90 ℃, the reagent (86) for washing the test substance (MD) may be affected by the temperature change and the alcohol may be vaporized. Therefore, the configuration in which the sealing body (30a) is unsealed after the nucleic acid amplification and the reagent (86) for washing the substance (MD) to be detected is transferred to the 2 nd chamber (12) is particularly effective in that the vaporization of the alcohol can be suppressed.

In the configuration further including the transfer mechanism (240), the control unit (230) preferably controls the rotation mechanism (220) so as to transfer the reagent (87) for denaturing nucleic acid from the 4 th liquid reservoir (34) provided in the cartridge (100) to the 3 rd chamber (13) connected to the 2 nd chamber (12) by rotation, and controls the transfer mechanism (240) so as to transfer the test substance (MD) accommodated in the 2 nd chamber (12) to the 3 rd chamber (13). With this configuration, in the cartridge (100), a treatment for denaturing nucleic acid can be performed on the nucleic acid after the treatment for washing nucleic acid in the 2 nd chamber (12). Furthermore, even when the treatment for denaturing nucleic acid is performed, since the cassette (100) can be transported to the 3 rd chamber (13) only by rotating the cassette around the rotation shaft (221) without using a liquid transfer medium such as air pressure, the treatment for denaturing nucleic acid can be performed with a simple apparatus configuration.

In this case, the control unit (230) preferably controls the unsealing mechanism (250) so as to unseal the closure body (30a) of the liquid reservoir unit (30) that contains the reagent (87) for denaturing the nucleic acid after the reaction of the substance (MD) to be detected and the reagent (81) is performed at least by the temperature adjustment unit (260). With this configuration, after amplification is performed by a temperature change using the temperature adjustment unit (260), the reagent (87) for denaturing nucleic acid is transferred to the 3 rd chamber (13), whereby the influence of the temperature change on the reagent (87) for denaturing nucleic acid can be suppressed.

In the configuration for transferring the substance to be detected (MD) stored in the 2 nd chamber (12) to the 3 rd chamber (13), the control unit (230) preferably controls the rotation mechanism (220) so as to transfer the reagent (88) containing the Labeling Substance (LS) that reacts with the amplification product of the nucleic acid from the 5 th liquid storage unit (35) provided in the cartridge (100) to the 4 th chamber (14) connected to the 3 rd chamber (13) by rotation, and controls the transfer mechanism (240) so as to transfer the substance to be detected (MD) stored in the 3 rd chamber (13) to the 4 th chamber (14). In such a configuration, the cartridge (100) may be subjected to not only a process of denaturing nucleic acids but also a process of labeling for detecting nucleic acids as a detection target substance (MD). Furthermore, even when the processing for labeling nucleic acids is performed, since the cartridge (100) can be transported to the 4 th chamber (14) only by rotating the cartridge by the rotating mechanism (220) without using a liquid transfer medium such as air pressure, the processing for labeling nucleic acids can be performed with a simple apparatus configuration.

In this case, the temperature adjustment unit (260) preferably amplifies the nucleic acid contained in the dispersoid in the chamber (11) by periodically changing the temperature of the cassette (100) over a plurality of temperature ranges, and then increases the temperature of the cassette (100) to react the nucleic acid in the 4 th chamber (14) with the Labeling Substance (LS). With this configuration, both the process of amplifying nucleic acid and the process of labeling nucleic acid can be performed in the cartridge (100) by the common temperature control unit (260). Therefore, not only the emulsion (83) but also the nucleic acid amplification treatment and the nucleic acid labeling treatment can be performed more easily with a simple apparatus configuration by using the cartridge (100).

In the configuration including the temperature adjustment unit (260), the temperature adjustment unit (260) is preferably configured to be relatively movable to a position away from a position in contact with the cartridge (100) disposed in the installation unit (210), and the temperature of the cartridge (100) is preferably changed in a state of contact with the cartridge (100). With this configuration, the temperature of the cartridge (100) can be easily changed because heat can be efficiently transferred by bringing the temperature adjusting unit (260) into contact with the cartridge (100) as compared with the case of heating the cartridge (100) in a non-contact manner.

In the configuration provided with the temperature control section (260), the substance (MD) to be detected is preferably a protein, and the reagent (81) contains a substrate that reacts with a labeling substance that specifically binds to the protein in the sample (80). With this configuration, when the substance to be detected (MD) is a protein, the reaction between the labeling substance for detecting the substance to be detected (MD) and the substrate can be surely performed in each droplet (84) while partitioning the substance to be detected (MD) in each droplet (84) contained in the emulsion (83).

The program according to claim 3 of the present invention functions as a control unit (230) that performs the following control by a computer connected to a sample processing apparatus that processes a sample (80) using a cartridge (100) having a chamber (11) for storing liquid: a cartridge (100) containing a sample (80) and a dispersant (82) in a chamber (11) is rotated around a rotation shaft (221) by a rotation mechanism (220) provided in a sample processing device, and the sample (80) and the dispersant (82) in the chamber (11) are stirred by rotating the cartridge (100), thereby forming an emulsion (83) in which a dispersoid containing the sample (80) is dispersed in the dispersant (82).

In the program according to claim 3, the cartridge (100) is rotated about the rotation shaft (221), and the inertial force accompanying the rotation of the cartridge (100) acts on the chamber (11) to stir the sample (80) and the dispersant (82) in the chamber (11). The dispersoid is finely sheared by the dispersant (82) with stirring. As a result, an emulsion (83) is formed in which droplets (84) containing a dispersoid of the substance (MD) to be detected in the sample (80) are dispersed in the dispersing agent (82). Thus, an emulsion (83) can be formed in the chamber (11) by rotating the cartridge (100) only about the rotation shaft (221) without using liquid feeding controlled by air pressure, and the emulsion (83) can be more easily prepared when a sample is processed using the cartridge.

The sample processing cartridge according to claim 4 of the present invention is provided with a sample introduction part (20) for introducing a sample (80) and a chamber (11) for storing the introduced sample (80) and a dispersant (82), and is configured to form an emulsion (83) in which a dispersoid containing the sample (80) is dispersed in the dispersant (82) by stirring the sample (80) and the dispersant (82) in the chamber (11) by rotating around a rotating shaft (221).

In the sample processing cartridge according to claim 4, the inertial force accompanying the rotation of the cartridge (100) acts on the chamber (11) by rotating the cartridge (100) about the rotation shaft (221), thereby stirring the sample (80) and the dispersant (82) in the chamber (11). The dispersoid is finely sheared by the dispersant (82) with stirring. As a result, an emulsion (83) is formed in which droplets (84) containing a dispersoid of the substance (MD) to be detected in the sample (80) are dispersed in the dispersing agent (82). Thus, an emulsion (83) can be formed in the chamber (11) by rotating the cartridge (100) only about the rotation shaft (221) without using liquid feeding controlled by air pressure, and the emulsion (83) can be more easily prepared when a sample is processed using the cartridge.

The sample processing cartridge according to claim 4 is preferably further provided with a liquid storage unit (30) that is fluidly connected to the chamber (11) and that stores a dispersant (82) in advance, and the dispersant (82) is preferably transferred from the liquid storage unit (30) to the chamber (11) by rotating about a rotating shaft (221). With this configuration, since the dispersant (82) is stored in advance, the operation of injecting the dispersant (82) into the cartridge (100) becomes unnecessary. Furthermore, the dispersant (82) can be transferred into the chamber (11) only by rotating the cartridge (100) about the rotating shaft (221) without using a liquid transfer controlled by air pressure. From these, when a sample is processed using the cartridge (100), the emulsion (83) can be more easily prepared.

[ Effect of the invention ]

When a cartridge is used to process a sample, it becomes possible to make an emulsion more easily.

[ brief description of the drawings ]

FIG. 1 is a view showing a sample treatment method before stirring (A) and after stirring (B).

Fig. 2 is a flow chart for explaining a sample processing method.

Fig. 3 is a schematic view showing the indoor part before (a) and after (B) stirring.

Fig. 4 is a schematic diagram showing an example (a) in which the rotation direction of the cassette is reversed and an example (B) in which the cassette is rotated in the same direction.

Fig. 5 is a side view (a) showing a mode of the structure of the sample processing apparatus and a view (B) of a cassette disposed in the setting section.

Fig. 6 is a schematic diagram for explaining the program.

Fig. 7 is a diagram showing an example in which a plurality of chambers are provided in a cassette.

Fig. 8 is a diagram showing an example in which a liquid storing unit is provided in a cartridge.

Fig. 9 is a diagram showing a specific configuration example of the cartridge.

FIG. 10 is a flowsheet illustrating an emulsion PCR assay.

FIG. 11 is a diagram for explaining the functions of each part of the cartridge in the emulsion PCR measurement.

FIG. 12 is a diagram illustrating the progress of the reaction in the emulsion PCR assay.

Fig. 13 is a plan view showing a mode of the example 1 of the temperature adjustment section.

Fig. 14 is a plan view showing a mode of the example 2 of the temperature adjustment section.

Fig. 15 is a perspective view showing a state where the cover of the sample processing apparatus is opened.

Fig. 16 is a perspective view showing a state where the cover of the sample processing apparatus is closed.

Fig. 17 is a sectional view showing a mode of an internal structure of the sample processing apparatus.

Fig. 18 is a block diagram showing a configuration for controlling the sample processing apparatus.

FIG. 19 is a schematic diagram showing an example of a mode of use of the sample processing apparatus.

FIG. 20 is a schematic diagram of a scattergram showing the results of nucleic acid detection according to the comparative example.

Fig. 21 is a view showing the rotation speed variation patterns (a) to (D) of the cartridge in example 1.

FIG. 22 is microscope images (A) to (D) of the emulsions in example 1.

Fig. 23 is schematic diagrams (a) to (D) of the scattergrams obtained in example 1.

FIG. 24 is a graph showing the results of comparison between the results of detection of nucleic acids in example 1 and those of comparative example.

FIG. 25 is schematic diagrams (A) to (D) showing chambers in the case of forming an emulsion at each volume ratio in example 2.

FIG. 26 is a graph showing the results of comparison between the results of detection of nucleic acids in example 2 and those of comparative example.

Fig. 27 is a graph showing a rotation speed variation pattern of the cartridge in example 3.

Fig. 28 is a schematic diagram showing the chambers at the time of emulsion formation in example 3.

FIG. 29 is a schematic diagram of a scattergram obtained in example 3.

Fig. 30 is a view showing the rotation speed variation patterns (a) to (D) of the cartridge in example 4.

Fig. 31 is schematic diagrams (a) to (D) of the scattergrams obtained in example 4.

FIG. 32 is a graph showing the results of comparison between the results of detection of nucleic acids in example 4 and those of comparative example.

Fig. 33 is a schematic diagram showing a cartridge (modification) used in immunoassay.

FIG. 34 is a diagram illustrating the progress of a reaction in an immunoassay.

Fig. 35 is a diagram for explaining the functions of each part of the cartridge in immunoassay.

Fig. 36 is a diagram for explaining the cartridge of embodiment 2.

Fig. 37 is a diagram showing a process flow using the cartridge of embodiment 2.

Fig. 38 is a diagram for explaining the cartridge of embodiment 3.

Fig. 39 is a diagram showing a process flow using a cartridge of embodiment 3.

Fig. 40 is a diagram showing a modification of the sample processing apparatus.

Fig. 41 is a diagram for explaining a conventional technique.

[ detailed description ] embodiments

[ embodiment 1 ]

Hereinafter, embodiments will be described based on the drawings.

(outline of sample processing method)

A sample processing method according to embodiment 1 will be described with reference to fig. 1 to 3. The sample processing method according to embodiment 1 is a sample processing method using a cartridge 100 having a chamber 11 for storing a liquid. The sample processing method is a method for performing a process for detecting a test substance MD using a cartridge 100 into which a sample 80 containing the test substance MD is injected.

The sample is, for example, a biological sample collected from a human as a subject. The sample is, for example, a liquid such as a body fluid or blood (whole blood, serum or plasma) collected from a patient, or a liquid obtained by subjecting a collected body fluid or blood to a predetermined pretreatment. The sample is a substance to be detected MD, and includes, for example, nucleic acids such as DNA (deoxyribonucleic acid), cellular and intracellular substances, proteins such as antigens and antibodies, and peptides. For example, when the substance to be detected MD is a nucleic acid, an extract solution obtained by extracting a nucleic acid from blood or the like by a predetermined pretreatment is injected into the cartridge 100 as the sample 80.

Further, the cartridge 100 may contain a reagent 81 for processing the detection target substance MD in the sample 80. The cartridge 100 contains the dispersant 82. The reagent 81 for treating the detection target substance MD reacts with another substance that binds to the detection target substance MD or the detection target substance MD. The number of the reagents 81 for treating the detection target substance MD may be 1 or more. The sample 80, the reagent 81 for treating the analyte MD, and the dispersing agent 82 are liquid. However, these liquids may contain other liquids. The dispersant 82 is immiscible with the sample 80 and the reagent 81. That is, the mixture of the sample 80 and the reagent 81 and the dispersant 82 have the property of being separated from each other. For example, the mixed liquid is aqueous, and the dispersant 82 is oil. Sample 80, reagent 81 and dispersant 82 are stirred in chamber 11 to form emulsion 83. That is, the mixed solution of the sample 80 and the reagent 81 and the dispersant 82 become a dispersion solution in which the dispersoid containing the sample 80 and the reagent 81 is dispersed as droplets 84 into the dispersant 82. In the present embodiment, the cartridge 100 may not contain the reagent 81. It is sufficient that an emulsion 83 is formed in which at least the dispersoid containing the sample 80 is dispersed in the dispersant 82.

The cartridge 100 is a replaceable consumable. That is, the cartridge 100 is discarded after being used for the measurement only a predetermined number of times. The usable number of the cartridge 100 is 1 or several times. The cartridge is a replaceable component that combines the functions necessary for processing the sample 80.

The cartridge 100 is, for example, a flat plate-shaped member forming an internal space. The cartridge 100 includes a chamber 11 capable of storing liquid therein. The chamber 11 is provided in 1 or more in the cartridge 100. The chamber 11 is configured as an internal space having a volume capable of accommodating the sample 80, the reagent 81, and the dispersant 82. The chamber 11 accommodates a sample 80, a reagent 81, and a dispersant 82. The cartridge 100 may include a space or the like functioning as a sample introduction part 20, a channel 40, a liquid storage part for introducing a liquid into the chamber 11. The cartridge 100 can form an internal space such as the chamber 11 or the passage by, for example, applying a transparent film to the surface of a member in which the hole constituting the chamber 11 or the passage 40 is formed to close the opening.

The chamber 11 may or may not contain the reagent 81 and the dispersant 82 in advance. In the chamber 11 not containing the reagent 81 and the dispersing agent 82, the reagent 81 and the dispersing agent 82 may be injected from another position in the cartridge 100 or from the outside of the cartridge 100.

As shown in fig. 2, the sample processing method of embodiment 1 includes the following steps S1 and S2. (S1) the sample 80 and the dispersing agent 82 are stored in the chamber 11 of the cartridge 100. (S2) the cartridge 100 is rotated about the rotation shaft 221 to stir the sample 80 and the dispersant 82 in the chamber 11, thereby forming an emulsion 83 in which the dispersoid containing the sample 80 is dispersed in the dispersant 82.

In step S1, the method of storing the liquid in the chamber 11 is not particularly limited. The operator can inject the sample 80, the dispersing agent 82, or the reagent 81 for treating the detection target substance MD in the sample 80 from the opening of the cartridge 100 by a technique using a pipette or the like. The liquid can be injected from the opening of the cartridge 100 by a dispensing device that dispenses the liquid. The sample 80 and the reagent 81 may be mixed in advance to be a mixed solution, and then injected into the cartridge 100.

In step S1, the sample 80, the reagent 81 for treating the test substance MD, and the dispersing agent 82 may be directly injected into the chamber 11. When the sample 80, the reagent 81 for processing the detection target substance MD, and the dispersing agent 82 are stored in a portion other than the chamber 11 of the cartridge 100, they are transferred to the chamber 11 through the passage 40 and the like. As a result of step S1, the sample 80, the reagent 81 for processing the test substance MD, and the dispersing agent 82 are contained in the same chamber 11.

In step S2, the cartridge 100 is rotated about the rotation shaft 221. The rotation axis 221 is an axis along a normal direction with respect to the surface of the flat plate-shaped cartridge 100. In order to suppress vibration accompanying rotation, the rotation shaft 221 is preferably disposed so as to be at a substantially center of gravity of the cartridge 100. In the case of the disk-shaped cartridge 100 as in fig. 1, the center of the circular cartridge 100 may also be regarded as the center of gravity.

At this time, the inertial force accompanying the rotation of the cartridge 100 acts on the chamber 11 to stir the sample 80, the reagent 81, and the dispersing agent 82 in the chamber 11. That is, the dispersoid including the sample 80 and the reagent 81 and the dispersant 82 move relatively in the chamber 11 by the inertial force. At this time, preferably, turbulence of the dispersoid and the dispersant 82 occurs in the chamber 11 by the rotation. Thereby facilitating agitation. With stirring, the dispersoid is finely sheared by the dispersant 82.

As a result, the dispersoid is finely divided in the dispersant 82, and a plurality of droplets 84 of the dispersoid partitioned by the interface between the dispersoid and the dispersant 82 are formed in the chamber 11. That is, an emulsion 83 is formed in which droplets 84 containing a dispersoid of the substance to be detected MD are dispersed in the dispersing agent 82. The droplets 84 contained in the emulsion 83 contain the substance to be detected MD. When the reagent 81 for processing the detection target substance MD is contained in the chamber 11, the droplet 84 contains the detection target substance MD and the reagent 81 for processing the detection target substance MD. At this time, the detection target substance MD can be reliably treated with the reagent 81 in each droplet 84 having a fine partition.

As described above, in the sample processing method according to embodiment 1, the emulsion 83 in which the droplets 84 of the dispersoid are dispersed in the dispersant 82 is formed by rotating the cartridge 100 about the rotation shaft 221. Thus, the emulsion 83 can be formed in the chamber 11 by simply rotating the cartridge 100 around the rotation shaft 221 without using liquid feeding controlled by air pressure, and the emulsion 83 can be more easily prepared when a sample is processed using the cartridge.

By reactive treatment of reagents

After the emulsion 83 is formed, the substance to be detected MD contained in the dispersoid can be reacted with the reagent 81. As shown in FIG. 3, in the emulsion 83, the substance to be detected MD and the reagent 81 are accommodated in each of the droplets 84 having a fine partition, and thus each of the substance to be detected MD having a partition can react with the reagent 81.

For example, after the emulsion 83 is formed, the substance to be detected MD contained in the dispersoid and the reagent 81 are reacted by changing the temperature of the cartridge 100. Thus, the substance to be detected MD and the reagent 81 for processing the substance to be detected MD can be reliably reacted by a temperature change in a state where the substance to be detected MD and the reagent 81 are accommodated in the droplets 84 of the dispersoid and partitioned. In addition, the substance MD and the reagent 81 can be efficiently reacted by a temperature change in the droplets 84 contained in the emulsion 83, as compared with the case where the reaction between the substance MD and the reagent 81 is naturally performed at the temperature of the installation environment of the cartridge 100.

(action of changing the rotational speed of the cartridge)

In the example shown in fig. 4(a) and (B), the emulsion 83 is formed in the chamber 11 by repeating an operation of changing the rotational speed of the cartridge 100 rotating about the rotational shaft 221. The rotation speed is changed to accelerate or decelerate the cartridge 100 rotating in a predetermined rotation direction, and the rotation direction of the cartridge 100 is reversed to rotate in the opposite direction, so that the cartridge is stopped by deceleration and accelerated in the opposite direction.

In this example, the rotational speed of the cartridge 100 rotating about the rotational axis 221 changes. The inertial force acting on the liquid in the chamber 11 varies with the change in the rotation speed. The dispersoid and the dispersant 82 in the stirring chamber 11 are stirred by the change of the inertial force. That is, the dispersoid and the dispersant 82 move relatively in the chamber 11 due to the change in the inertial force caused by the change in the rotation speed. Thereby, the stirring is performed quickly. In this case, it is preferable that turbulence of the dispersoid and the dispersant 82 occurs in the chamber 11 due to a change in the rotation speed. Thereby facilitating agitation.

Further, the operation of changing the rotation speed of the cartridge 100 rotating around the rotation shaft 221 is repeated. The operation of changing the rotation speed of the cartridge 100 is repeated, for example, a predetermined number of times. The repetition of the operation of changing the rotational speed of the cartridge 100 may be performed at a constant cycle, for example, or may be repeated for a predetermined time period after the operation of changing the rotational speed is started. As shown in fig. 3, the stirring is efficiently performed in the chamber 11 as a result of repeated changes in the rotation speed. The dispersoid is finely sheared by the dispersant 82 by stirring.

In this way, by repeating the operation of changing the rotational speed of the cartridge 100, the inertial force accompanying the speed change acts to efficiently stir the sample 80 and the dispersant 82 in the chamber 11, and the dispersoid can be rapidly finely sheared by the dispersant 82. As a result, the emulsion 83 can be efficiently produced.

The operation of changing the rotational speed of the cartridge 100 includes an operation of reversing the rotational direction of the cartridge 100 or an operation of accelerating and decelerating in the same direction.

Fig. 4(a) shows an example of an operation of reversing the rotation direction of the cartridge 100. For example, the cartridge 100 rotates alternately to one side and the other side around the rotation shaft 221 with the rotation shaft 221 as the center. At this time, the rotation speed is positive (+) at one side and negative (-) at the other side about the rotation axis 221, and is positive, 0, negative, 0, positive …, and the acceleration from the stop state and the deceleration to the stop state are repeated every time the rotation direction is reversed. This can apply a large inertial force to the liquid in the chamber 11. As a result, the dispersoid and the dispersant 82 are efficiently stirred. In this way, when the rotation direction of the cartridge 100 is reversed, a large inertial force can be applied when the rotation direction is reversed, and the emulsion 83 can be efficiently produced.

Fig. 4(B) shows an example of the operation of accelerating and decelerating in the same direction. In fig. 4(B), acceleration is inexpensively represented by a solid arrow in the circumferential direction of the cartridge 100, and deceleration is represented by a broken arrow. In fig. 4(B), the cartridge 100 is rotated in a clock rotation direction, or may be rotated in a counterclockwise direction. The cartridge 100 rotates around the rotation shaft 221 so as to alternately repeat acceleration and deceleration in a predetermined direction around the rotation shaft 221. The deceleration may be to stop the rotation of the cartridge 100, or may be to repeat the acceleration and deceleration between a designated high speed and a designated low speed. In fig. 4(B), the acceleration and deceleration are alternately repeated, and the cartridge 100 may also be rotated in a pattern that is not alternately repeated, such as, for example, acceleration, deceleration, acceleration, deceleration …. Since there is no need to reversely rotate a rotation mechanism for rotating the cartridge 100 when accelerating and decelerating the cartridge 100 in the same direction, the emulsion 83 can be easily prepared.

(outline of sample processing apparatus)

Next, an outline of the sample processing apparatus 200 according to embodiment 1 will be described with reference to fig. 5.

The sample processing apparatus 200 according to embodiment 1 is an apparatus for carrying out the above-described sample processing method. That is, the sample processing apparatus 200 is an apparatus for processing the detection object MD contained in the sample using the cartridge 100 including the chamber 11 for storing the liquid. The mixing, stirring, heating or cooling of the dispersoids containing the sample 80 and the dispersant 82, the movement of the solid or liquid used for the treatment, and other various operations can be performed in the cartridge 100 by the sample treatment apparatus 200.

As shown in fig. 5(a), the sample processing apparatus 200 includes an installation unit 210, a rotation mechanism 220, and a control unit 230. The installation unit 210, the rotation mechanism 220, and the control unit 230 are housed in the housing 201, for example.

The housing 201 is formed of a box-shaped member having an internal space with a predetermined volume, a combination of a frame and an exterior plate, or the like. For example, the housing 201 of the sample processing device 200 has a small box-like shape that can be set on a stage.

The setting unit 210 can set the cartridge 100 having the chamber 11 for storing the sample 80 and the dispersing agent 82. The setting unit 210 is, for example, an upper surface constituting a setting surface of the cartridge 100 and supports a lower surface of the cartridge 100. The setting section 210 supports the cartridge 100 rotatably about a rotation shaft 221. In the example of fig. 5(a), the setting part 210 itself is provided to the rotating shaft 221 so as to be rotatable integrally with the rotating shaft 221. The installation portion 210 is provided separately from the rotation shaft 221, for example, and may rotatably support the cartridge 100 via a bearing or the like.

The rotation mechanism 220 can rotate the cartridge 100 set in the setting section 210 about the rotation shaft 221. The rotating mechanism 220 may include a rotating shaft 221 and a driving part 222 that rotates the rotating shaft 221. The rotation axis 221 extends, for example, in the vertical direction. In the example of fig. 5(a), the rotation mechanism 220 supports the installation portion 210 fixed to one end of the rotation shaft 221, and the rotation shaft 221 and the installation portion 210 are rotated integrally by the driving portion 222 connected to the other end of the rotation shaft 221. The driving unit 222 is, for example, an electric motor. Thus, the rotation mechanism 220 rotates the cartridge 100 placed on the setting section 210 in the horizontal plane about the rotation shaft 221. The rotating mechanism 220 may change the rotation speed of the rotating shaft 221 to at least 2 stages. The at least 2 stages may also be stopped as 2 stages of rotation speed 0, stopping and rotation, for example.

The control unit 230 can control the rotating mechanism 220. Specifically, the controller 230 performs control of stirring the sample 80, the reagent 81, and the dispersant 82 in the chamber 11 by rotating the cartridge 100 containing the sample 80 and the dispersant 82 in the chamber 11 by the rotating mechanism 220, thereby forming the emulsion 83 in which the dispersoid containing the sample 80 is dispersed in the dispersant 82. As a result, the controller 230 forms an emulsion 83 (see fig. 5B) in which the dispersoid containing the sample 80 is dispersed in the dispersant 82 in the chamber 11. The control unit 230 is constituted by a computer including a processor such as a CPU and a storage unit such as a memory. The control unit 230 may be configured by a programmable device such as an FPGA (field-programmable gate array) configured for the sample processing device 200.

The sample processing apparatus 200 can form the emulsion 83 by performing the sample processing method shown in fig. 2 with such a configuration. That is, in the sample processing apparatus 200, when the cartridge 100 containing the sample 80 and the dispersing agent 82 in the chamber 11 is set in the setting unit 210, the control unit 230 controls the rotating mechanism 220 to rotate the cartridge 100 about the rotating shaft 221. As a result, the sample processing apparatus 200 stirs the sample 80 and the dispersant 82 in the chamber 11 to form the emulsion 83 in the chamber 11 of the cartridge 100. As will be described later, the sample processing apparatus 200 may be configured to accommodate the sample 80, the reagent 81, and the dispersing agent 82, which are disposed at positions other than the chamber 11 in the cartridge 100, in the chamber 11. The sample processing apparatus 200 may form an emulsion 83 in which a dispersoid including the sample 80 and the reagent 81 is dispersed in the dispersing agent 82, and perform a process of reacting the analyte MD with the reagent 81 in each of the finely divided droplets 84.

In this way, in the sample processing apparatus 200 according to embodiment 1, the emulsion 83 can be formed in the chamber 11 only by rotating the cartridge 100 about the rotation shaft 221 without using the liquid feeding controlled by the air pressure. As a result, when the cartridge is used to process a sample, it becomes possible to make the emulsion 83 more easily. Further, when the emulsion is formed by using the liquid feeding controlled by the air pressure, it is necessary to provide a mechanism for adding the air pressure to each of the portion for storing the dispersing agent 82 and the portion for storing the sample, or a mechanism for precisely controlling the air pressure, and in the configuration shown in fig. 5, the emulsion 83 can be formed only by providing the rotating mechanism 220. Therefore, the apparatus configuration can be simplified, and a smaller and simpler sample processing apparatus 200 can be obtained.

As shown in fig. 4(a) and 4(B), the controller 230 may control the rotating mechanism 220 to repeat the operation of changing the rotational speed of the cartridge 100 rotating about the rotating shaft 221, thereby forming the emulsion 83 in the chamber 11. In such a configuration, by repeating the operation of changing the rotational speed of the cartridge 100, the inertial force accompanying the speed change acts to effectively stir the sample 80, the reagent 81, and the dispersant 82 in the chamber 11, and the dispersoid can be rapidly finely sheared by the dispersant 82. As a result, the emulsion 83 can be efficiently produced.

(procedure)

Next, a description will be given of a program 300 according to embodiment 1 with reference to fig. 6.

As shown in fig. 6, the sample processing apparatus 200 may not include the control unit 230 and the rotation mechanism 220 may be controlled by an external computer 310. The program 300 according to embodiment 1 is a control program for causing the computer 310 to function as a control unit of the sample processing apparatus 200.

The computer 310 includes, for example, a processor 311 such as a CPU, a storage unit 312 including a volatile memory, a nonvolatile memory, and the like, and an input/output unit 313 connected to an external device or a network including the sample processing apparatus 200 by wire or wireless. The program 300 is stored in the storage 312 and executed by the processor 311, whereby the computer 310 functions as a control unit of the sample processing apparatus 200.

That is, the program 300 functions as the control unit 230 that performs the following control by a computer connected to a sample processing apparatus that processes the sample 80 using the cartridge 100 including the chamber 11 for storing the liquid: the cartridge 100 containing the sample 80 and the dispersant 82 in the chamber 11 is rotated around the rotation shaft 221 by the rotation mechanism 220 provided in the sample processing apparatus, and the sample 80 and the dispersant 82 in the chamber 11 are stirred by rotating the cartridge 100, thereby forming the emulsion 83 in which the dispersoid containing the sample 80 is dispersed in the dispersant 82.

The program 300 may be provided, for example, by recording a non-transitory (non-transient) recording medium 320 that is read by a computer. The program 300 recorded in the recording medium 320 is read by a reading device, not shown, of the computer 310 and stored in the storage unit 312 of the computer 310. The recording medium 320 does not include a transient propagation signal for transmitting the program 300, and includes, for example, a nonvolatile semiconductor memory, an optical or magnetic recording medium, and the like. Additionally, process 300 may be provided, for example, by a transitory, propagating signal conveying process 300, from a network 330 connected to computer 310.

In the program 300 of embodiment 1, the computer 310 functions as the control unit 230 that performs the following control, as described above, to form the emulsion 83 in which the dispersoid containing the sample 80 is dispersed in the dispersing agent 82 by stirring the sample 80 and the dispersing agent 82 in the chamber 11 by rotating the cartridge 100, and to form the emulsion 83 in the chamber 11 by rotating the cartridge 100 only about the rotating shaft 221 without using the liquid feeding controlled by the air pressure. As a result, when the cartridge is used to process a sample, it becomes possible to make the emulsion 83 more easily.

(sample processing box)

Next, a sample process cartridge (hereinafter, referred to as a cartridge 100) according to embodiment 1 will be described with reference to fig. 1.

The cartridge 100 includes a sample introduction portion 20 for introducing a sample 80. The sample introduction portion 20 is, for example, an opening formed in the outer surface of the cartridge 100. The opening of the sample introduction section 20 may be plugged in advance, and the cartridge 100 may be unsealed by an operator when used. The sample introduction section 20 accommodates at least a sample 80. The sample introduction section 20 can accommodate a mixed solution of a sample 80 and a reagent 81 for treating the detection target substance MD.

The cartridge 100 includes a chamber 11 capable of storing liquid therein. The chamber 11 may be a space that is substantially closed so that liquid does not leak out inside the cartridge 100. The substantially closed space is a space that allows communication with the outside of the cartridge 100 through the sample introduction portion 20. The chamber 11 may be configured such that the liquid in the chamber 11 does not flow back to the sample introduction part 20 under normal use conditions. The chamber 11 can accommodate the introduced sample 80, a reagent 81 for treating the detection target substance MD in the sample 80, and a dispersing agent 82.

The cartridge 100 is configured to stir the sample 80 and the dispersant 82 in the chamber 11 by rotating around the rotation shaft 221, thereby forming an emulsion 83 in which a dispersoid containing the sample 80 is dispersed in the dispersant 82. The cartridge 100 may be configured to form the emulsion 83 in the chamber 11 by repeating an operation of changing the rotation speed when rotating around the rotation shaft 221.

As described above, the cartridge 100 is, for example, a flat plate-shaped member forming an internal space. The case 100 may have a shape without a flat plate, a block shape, a ball shape, or the like. Since the dispersoid and the dispersant 82 in the chamber 11 are stirred by the inertial force when the rotation speed is changed, it is preferable that acceleration and deceleration can be easily performed, the cartridge 100 is lightweight, does not become eccentric so as not to vibrate with rotation, and is more specifically preferable to be disk-shaped.

The shape of the chamber 11 formed in the cartridge 100 is not particularly limited, and preferably has a shape extending in the circumferential direction around the rotation axis 221 or in the tangential direction of a circle centered on the rotation axis 221. When the dispersoid containing, for example, the sample 80 and the reagent 81 is an aqueous system and the dispersant 82 is an oil-based liquid, the dispersoid and the dispersant 82 are separated vertically in the chamber 11 as shown in FIG. 3. Therefore, when the dispersoid and the dispersing agent 82 are stirred by changing the rotation speed, the shearing force is easily applied between the dispersoid and the dispersing agent 82 as the area of the interface where the dispersoid and the dispersing agent 82 contact each other is increased. Therefore, the chamber 11 is preferably flat in the direction of rotation. Further, when the chamber 11 is oriented in the radial direction around the rotation shaft 221, the inertial force is likely to vary between the inner and outer circumferential sides in the radial direction due to the change in the rotation speed. Therefore, the chamber 11 preferably extends in the circumferential direction around the rotation axis 221 or in the tangential direction of a circle centered on the rotation axis 221.

In the cartridge 100 according to embodiment 1, when the cartridge 100 is rotated about the rotation shaft 221, an inertial force accompanying the rotation of the cartridge 100 acts on the chamber 11 to stir the sample 80 and the dispersing agent 82 in the chamber 11. With stirring, the dispersoid is finely sheared by the dispersant 82. As a result, an emulsion 83 is formed in which droplets 84 containing the dispersoid of the substance to be detected MD are dispersed in the dispersing agent 82. Thus, the emulsion 83 can be formed in the chamber 11 by simply rotating the cartridge 100 around the rotation shaft 221 without using liquid feeding controlled by air pressure, and the emulsion 83 can be more easily prepared when a sample is processed using the cartridge.

In the example shown in fig. 7, the cartridge 100 contains a plurality of chambers 11. That is, a plurality of chambers 11 for forming the emulsion 83 in which the dispersoid containing the sample 80 is dispersed in the dispersing agent 82 are provided in the cartridge 100. Thus, a plurality of samples 80 collected from different living bodies or the same sample 80 in an amount exceeding the processing amount of 1 chamber 11 can be collectively processed in 1 cartridge 100 to form the emulsion 83.

In the example of fig. 7, the cartridge 100 includes a plurality of chambers 11 arranged in an arc shape having substantially the same distance from the radial direction of the rotation axis 221. In fig. 7, the plurality of chambers 11 are provided at positions where the rotation loci of the respective chambers 11 overlap each other in a plan view. The circumferential movement speed of the chamber 11 when the cartridge 100 is rotated is proportional to the radial distance D1 from the rotation axis 221 of each chamber 11. Therefore, when the distances from the rotation shafts 221 of the respective chambers 11 are different, the magnitudes of the inertial forces acting on the respective chambers 11 are different, and the quality of the produced emulsion 83 may be disturbed for each chamber 11. In contrast, in fig. 7, since the plurality of chambers 11 are arranged in an arc shape having substantially the same radial distance from the rotation axis 221, the magnitude of the inertial force acting on each chamber 11 with rotation becomes substantially equal, and the quality of the emulsion 83 to be produced can be made more uniform. For example, the size of the droplets 84 contained in the emulsion 83 produced in each chamber 11 or the number of droplets 84 can be suppressed from being scattered by the rotation.

As shown in fig. 8, the cartridge 100 may further include a liquid storage unit 30 for storing a liquid used for a process, in addition to the sample introduction unit 20 and the chamber 11. The liquid storage unit 30 is a space formed to have a predetermined volume in the cartridge 100, similarly to the chamber 11. The liquid storage portion 30 is connected to the chamber 11 through a passage 40 formed in the cartridge 100.

Fig. 8 shows a liquid storage unit 30 in which a dispersant 82 is stored in advance as an example of the liquid storage unit 30. That is, the cartridge 100 includes the liquid storage portion 30 that is fluidly connected to the chamber 11 and that stores the dispersant 82 in advance. Further, the cartridge 100 is configured to transfer the dispersant 82 from the liquid storage unit 30 to the chamber 11 by rotating about the rotation shaft 221.

Specifically, the liquid storage unit 30 is provided in the cartridge 100 at a position closer to the rotation shaft 221 than the chamber 11. The passage 40 is provided in the liquid storage unit 30 at a position on the outer circumferential side in the radial direction around the rotation shaft 221, and is connected to the chamber 11. Therefore, when the cartridge 100 rotates about the rotation shaft 221, a centrifugal force radially outward acts on the dispersant 82 stored in the liquid storage unit 30. As a result, the dispersant 82 is transferred from the liquid storage unit 30 into the chamber 11 by the centrifugal force generated by the rotation. When the liquid is transferred from the liquid storage unit 30 into the chamber 11, a centrifugal force of a magnitude necessary for moving the liquid may be generated, and thus the rotational speed of the cartridge 100 does not need to be repeatedly changed.

With this configuration, since the dispersing agent 82 is stored in the cartridge 100 in advance, the operation of injecting the dispersing agent 82 into the cartridge 100 becomes unnecessary. Further, the dispersant 82 can be transferred into the chamber 11 only by rotating the cartridge 100 about the rotation shaft 221 without using a liquid transfer controlled by air pressure. From these, the emulsion 83 can be more easily prepared when the cartridge 100 is used to process a sample

(specific construction example of sample processing method)

Next, a specific configuration example of the sample processing method according to embodiment 1 will be described. An example using the cartridge 100 shown in fig. 9 will be described below.

Box (a)

Specific configuration examples of the cartridge 100 will be described. In the example of fig. 9, the cartridge 100 is a disk-shaped cartridge configured by a plate-shaped and disk-shaped substrate 50. Each part in the cartridge 100 is formed by bonding a through hole formed in the substrate 50 and a film, not shown, on both surfaces of the substrate 50, covering the entire surface including the through hole. The substrate 50 has a thickness that facilitates temperature adjustment of the cartridge 100 by a temperature adjustment unit 260 (see fig. 17) to be described later. For example, the thickness of the substrate 50 is set to several mm, specifically, about 1.2 mm.

The substrate 50 is provided with a hole 51 and a sample processing region 52. The sample processing area 52 is provided with a plurality of chambers 11 to 16, a sample introduction part 20, a plurality of liquid storage parts 30, and a plurality of passages 40.

The hole 51 penetrates the substrate 50 at the center of the substrate 50. The cartridge 100 is provided in the sample processing device 200 such that the center of the hole 51 coincides with the center of the rotation shaft 221. Hereinafter, the radial direction and the circumferential direction of a circle having the hole 51 as the center are referred to as the "radial direction" and the "circumferential direction", respectively.

The sample introduction portion 20 is an opening for introducing the sample 80 into the cartridge 100. In the example of fig. 9, a mixture of a sample 80 and a reagent 81 for treating a test substance MD is injected into the sample introduction portion 20. The mixed solution is previously injected into the cartridge 100 created by the operator, but the sample 80 and the reagent 81 injected separately may be mixed in the cartridge 100.

The sample 80 is a solution containing nucleic acid as the test substance MD. The nucleic acid is, for example, DNA of the subject. This enables formation of an emulsion 83 (see fig. 3) in which fine droplets 84 of a dispersoid containing a nucleic acid as a test substance MD and a reagent 81 for treating the test substance MD are dispersed in a dispersing agent 82. As a result, by partitioning each detection target substance MD within the droplet 84, each detection target substance MD can be reliably treated with the reagent 81.

The reagent 81 contains a nucleic acid amplification reagent for amplifying the nucleic acid in the sample 80. The nucleic acid amplification reagent contains a substance necessary for PCR such as DNA polymerase. The reagent 81 contains magnetic particles PM that bind to the nucleic acids in the sample 80 (see fig. 12). The magnetic particles PM provide a primer for nucleic acid amplification to the surface. The magnetic particles include a material having magnetic properties as a base material, and any particles may be used as long as they are used for ordinary measurement of a specimen. For example, Fe can be used as the base material₂O₃And/or Fe₃O₄Magnetic particles of cobalt, nickel, phyllite, magnetite, etc. The sample 80, the nucleic acid amplification reagent, and the magnetic particles PM mixed in the dispersion are introduced into the cartridge 100 from the sample introduction unit 20. In the dispersoid, a nucleic acid as a detection target substance MD is bound to the magnetic particles PM.

The chambers 11 to 16 are chambers capable of storing liquid. The chambers 11-16 are arranged in the circumferential direction near the outer periphery of the substrate 50. FIG. 9 shows an example in which 6 chambers 11 to 16 are provided in a cartridge 100.

The liquid storage unit 30 is a storage space for storing the liquid transferred to the chamber. The liquid storage portions 30 are connected to each other via any chamber and the passage 40. Fig. 9 shows an example in which 9 liquid storage units 30 are provided in the cartridge 100. The liquid storage units 30 are arranged in the circumferential direction near the center of the substrate 50. The liquid storage units 30 are each disposed closer to the rotation shaft 221 than the connected chambers. Therefore, by rotating the cartridge 100 about the rotation shaft 221, the liquid stored in the liquid storage unit 30 can be transferred from the liquid storage unit 30 to the chamber by centrifugal force.

The liquid storage portion 30 includes a closing body 30a closing a connection portion with the chamber 11. The liquids in the liquid storage portions 30 are stored in the liquid storage portions 30 in advance when the cartridge 100 is manufactured. The closing body 30a is a plug that closes the liquid storage section 30 and the external space. The sealing body 30a is configured to be openable by pressing the plug from above by an unsealing mechanism 250 (see fig. 17) of the sample processing apparatus 200. Before the closing body 30a is opened, the reagent in the liquid storage portion 30 does not flow through the passage 40, and when the closing body 30a is opened, the reagent in the liquid storage portion 30 flows out.

The liquid reservoir 30 includes a sample reservoir 38 for storing the mixed solution injected from the sample introduction unit 20. By rotating the cartridge 100 about the rotation shaft 221, the mixed solution of the sample 80 and the reagent 81 is transferred from the sample storage portion 38 to the chamber 11.

The liquid storing parts 30 storing the reagents in advance store the reagents capable of performing the measurement for 1 time. That is, the cartridge 100 includes a liquid storage unit 30 that stores a reagent capable of measuring 1 minute of a substance to be detected. In the cartridge 100 configured as described above, each cartridge 100 contains a reagent intended to be used up 1 time.

In fig. 9, 6 chambers 11, 12, 13, 14, 15, and 16 having substantially the same shape are arranged adjacent to each other in the circumferential direction, and are connected to a passage 40 extending in the circumferential direction. Between these 6 chambers 11 to 16, the substance to be detected is transferred 1 by 1 from one side (chamber 11 side) to the other side (6 th chamber 16 side) through the passage 40.

The passage 40 includes 6 radial direction regions 41 extending in the radial direction and an arc-shaped circumferential direction region 42 extending in the circumferential direction. The circumferential direction region 42 is associated with 6 radial direction regions 41. The 6 radial regions 41 are associated with the respective chambers 11 to 16. The respective liquid storage portions 30 are associated with chambers or passages through flow paths 43 extending substantially in the radial direction.

The detection target substance MD bonded to the magnetic particles PM is transferred to the passage 40 and the other chambers 11 by a combination of rotation of the cartridge 100 and action of magnetic force. Specifically, the magnetic particles PM move in the radial direction between the inside of the chamber 11 and the arc-shaped circumferential region 42 of the passage 40 by magnetic force. By rotating the cartridge 100, the magnetic particles PM move in the circumferential direction in the arc-shaped circumferential direction region 42. When the magnetic particles PM move to the position of the adjacent chamber, the magnetic particles PM move in the radial direction by the magnetic force between the arc-shaped circumferential direction region 42 of the passage 40 and the inside of the adjacent chamber. In this way, the magnetic particles PM bound to the detection object MD move sequentially toward the chambers 11 to 16 by a combination of radial movement by the action of magnetic force and circumferential movement by rotation.

In the example of fig. 9, the sample processing region 52 is formed only in a region of approximately 1/3 of the substrate 50. However, the present invention is not limited to this, and 2 sample processing regions 52 may be formed in 2 out of the remaining 3 regions of the substrate 50, and 3 sample processing regions 52 may be provided on the substrate 50. Further, the 1 sample processing region 52 may be formed over a region larger than 1/3 of the substrate 50.

In addition, the number and shape of the chambers 11 and the passages 40 are not limited to those shown in fig. 9. The configuration of each part of the sample processing region 52 is determined in accordance with the content of the sample processing measurement performed in the sample processing region 52.

Flow of sample treatment

Next, an example of specific measurement using the cartridge 100 of fig. 9 will be described.

FIG. 10 shows an example of the flow of the emulsion PCR assay. FIG. 12 is a diagram illustrating the progress of the reaction in the emulsion PCR measurement. The process using the cartridge 100 shown in fig. 9 may also include steps S13 to S19, and further include step S20.

In step S11, DNA is extracted from a sample such as blood by pretreatment (see fig. 12 a). The pretreatment is carried out using a dedicated nucleic acid extraction apparatus.

In step S12, the extracted DNA is amplified by the Pre-PCR process (see FIG. 12 (A)). The Pre-PCR treatment is a treatment for preliminarily amplifying DNA contained in the pretreated extract liquid to such an extent that the subsequent emulsion preparation treatment becomes possible. In the Pre-PCR treatment, the extracted DNA and a reagent for PCR amplification containing a polymerase or a primer are mixed, and the DNA in the dispersoid is amplified under temperature control using a thermal cycler. The thermal cycler performs 1 cycle thermal cycle processing in which the dispersoids are repeatedly changed to a plurality of different temperatures a plurality of times. The Pre-PCR treatment may be omitted depending on the concentration of the extracted nucleic acid.

Step S13 is an emulsion forming step of dispersing a dispersion containing a nucleic acid (DNA) as a test substance MD, a nucleic acid amplification reagent, and magnetic particles PM serving as a carrier for the nucleic acid in the dispersing agent 82. The reagent used for the amplification reaction of nucleic acid contains a substance necessary for PCR such as DNA polymerase. In step S13, an emulsion 83 containing a reagent including magnetic particles, polymerase, or the like and DNA is formed using the cartridge 100 (see fig. 12B). That is, droplets 84 containing a nucleic acid amplification reagent such as magnetic particles PM or polymerase and a DNA-containing dispersoid are formed, and a dispersoid composed of a large number of droplets 84 is dispersed in the dispersing agent 82. As a result, the DNA as the test substance MD is dispersed in the minute compartments in a limited dilution (the target component is diluted to 1 or 0 in each minute compartment). That is, each droplet 84 in the emulsion 83 is a minute partition, and each droplet 84 is formed so as to contain about 1 magnetic particle and a target DNA molecule as a detection target substance MD in the droplet 84.

Step S14 is an emulsion PCR step of amplifying the nucleic acid (DNA) in the droplets 84 formed in the emulsion forming step. In step S14, the DNA is bound to the primers on the magnetic particles PM in each droplet 84 of the emulsion 83 by temperature control using a thermal cycler, and amplified (emulsion PCR) (see fig. 12C). Thereby, the target DNA molecule is amplified in each droplet 84. That is, an amplification product of the nucleic acid is formed within each droplet 84. The amplified nucleic acid is bound to the magnetic particles PM as a carrier within the droplet 84 via the primer.

Step S15 is an emulsion breaking step of breaking droplets 84 containing magnetic particles PM carrying an amplification product of nucleic acid (DNA) obtained in the emulsion PCR step. In other words, step S15 is a step of demulsifying the emulsion 83 after the emulsion PCR step. After the DNA is amplified on the magnetic particles PM in step S14, the emulsion 83 is broken and the magnetic particles PM containing the amplified DNA are taken out from the droplet 84 (emulsion breaking) in step S15. For breaking the emulsion, a reagent 85 (see fig. 11) for demulsifying the emulsion is used. The agent 85 for demulsifying the emulsion comprises alcohol, a surfactant, or the like.

Step S16 is a cleaning step of cleaning the magnetic particles PM taken out of the droplets 84 by demulsification in the emulsion breaking step. In step S16, the magnetic particles PM taken out of the liquid droplets 84 are washed (first washing) with a reagent 86 (see fig. 11) for washing the detection object MD. The reagent 86 for washing the test substance MD is, for example, alcohol. The alcohol removes the oil film on the magnetic particles.

Step S17 is a denaturing step of denaturing the amplified double-stranded DNA bound to the magnetic particles PM into single strands. In step S17, the washed magnetic particles PM are denatured by the reagent 87 (see fig. 11) for denaturing nucleic acid. The reagent 87 for denaturing nucleic acid changes from pH to denature the amplified double-stranded DNA into single strands. The reagent 87 for denaturing nucleic acid contains an alkaline reagent such as an aqueous sodium hydroxide solution.

Step S18 is a hybridization step for reacting the amplified product of DNA with a labeling substance. After the denaturation step, in step S18, the DNA denatured into single strands on the magnetic particles PM is hybridized (hybridized) with the labeling substance LS by the reagent 88 (see fig. 11) containing the labeling substance LS reacted with the amplification product of nucleic acid (see fig. 12D). The labeling substance LS contains, for example, a fluorescent substance. The labeling substance LS is designed to specifically bind to the DNA of the detection target.

In step S19, the magnetic particles PM carrying the DNA bound to the labeling substance LS are washed (the 2 nd washing) with the reagent 89 (see fig. 11) for washing the detection target substance MD. In the 2 nd washing step, PBS (phosphate buffered saline) is used as a washing solution, for example. The PBS removed unreacted labeling substance not bound to the DNA (including labeling substance non-specifically adsorbed to the magnetic particles).

In step S20, the hybridized labeling substance LS detects DNA as the detection target substance MD. The DNA is detected, for example, by flow cytometry. In the flow cytometer, magnetic particles containing DNA bound to a labeling substance are passed through a flow cell, and laser light is irradiated to the magnetic particles. Fluorescence of the labeling substance emitted by the irradiated laser is detected.

DNA can also be detected by image processing. For example, magnetic particles containing DNA bound to a labeling substance are dispersed on a flat glass slide, and the dispersed magnetic particles are imaged by an imaging unit. Based on the captured image, the number of fluorescent magnetic particles is counted. As described above, in the sample processing method of the present embodiment, the detection target substance MD may be treated with the reagent 88 containing the labeling substance LS for labeling the detection target substance MD contained in the sample 80, and the signal based on the labeling substance LS may be detected. The sample processing device 200 of the present embodiment may further include a detection unit for detecting a signal based on the label substance LS.

Next, a process using the cartridge 100 will be described in detail with reference to fig. 11.

The cartridge 100 contains a chamber 11 in which an emulsion 83 is formed. As shown in FIG. 11, steps S13-S15 are performed in chamber 11.

The liquid storage section 30 includes a 1 st liquid storage section 31 for storing the dispersant 82. The dispersant 82 contains an oil immiscible with the sample 80 and the reagent 81. Thus, in this example, the dispersoid is aqueous and the dispersant 82 is oil. The dispersant 82 is stored in the 1 st liquid storage unit 31 in advance when the cartridge 100 is manufactured.

Emulsion formation

In step S13, first, a dispersion containing a nucleic acid (DNA) as a test substance MD, a nucleic acid amplification reagent, and magnetic particles PM as a carrier for the nucleic acid is injected from the sample introduction unit 20 into the sample storage unit 38. Further, the closure 30a of the 1 st liquid storage part 31 is unsealed. Further, the cartridge 100 rotates about the rotation shaft 221. The dispersant 82 is transferred from the 1 st liquid reservoir 31 to the chamber 11 by centrifugal force accompanying rotation, and the dispersoid is transferred from the sample reservoir 38. Thus, the operation of storing the dispersant 82 in the chamber 11, instead of just generating the emulsion 83, may be performed only by rotating the cartridge 100. Therefore, for example, since air pressure is not required to be added to the 1 st liquid storage part 31, the sample can be processed more easily.

The chamber 11 accommodates a sample 80 to be introduced, a reagent 81 for treating a substance to be detected MD in the sample 80, and a dispersing agent 82. Further, the cartridge 100 repeats the operation of changing the rotation speed while rotating about the rotation shaft 221, thereby forming an emulsion 83 in which a dispersoid including the sample 80 and the reagent 81 is dispersed in the dispersing agent 82 in the chamber 11.

An emulsion 83 containing droplets 84 of a dispersoid containing 1 molecule or 1 detection target substance MD is formed in the chamber 11 by the rotation of the cartridge 100. As a result, as shown in fig. 12, 1 test substance MD contained in the sample can be reliably handled by being partitioned in each droplet 84 contained in the emulsion 83. As a result, since the sample can be processed with high accuracy even if the sample contains only a very small amount of the analyte MD, the detection accuracy can be improved when the processed analyte MD is detected.

Emulsion PCR

In step S14, after the emulsion 83 is formed, the nucleic acid contained in the dispersoid is amplified by periodically changing the temperature of the cartridge 100 over a plurality of temperature ranges. Thus, the thermal cycle treatment can be performed on the droplets 84 (see fig. 12) contained in the emulsion 83 to efficiently amplify the nucleic acid.

The thermal cycle is performed in the chamber 11 by the temperature adjustment unit 260 (see fig. 13) provided in the sample processing apparatus 200. The temperature adjusting portion 260 is constituted by at least a device that changes the temperature of the cartridge 100 by heating. The temperature adjusting unit 260 may be constituted by a heating device such as a heater or a device capable of heating and cooling such as a Peltier element, for example. In step S14, the temperature of the cartridge 100 is changed by bringing the temperature adjustment portion 260 into contact with the cartridge 100. Thus, when the cartridge 100 is heated in a non-contact manner, the temperature adjustment unit 260 is brought into contact with the cartridge 100, so that heat can be efficiently transferred, and the temperature of the cartridge 100 can be easily changed.

The temperature adjusting portion 260 is in contact with the disk-shaped cartridge 100 from one or both of the upper surface and the lower surface of the cartridge 100 to adjust the temperature. For example, in fig. 13, a temperature adjustment portion 260 having a shape along a circumferential direction around a rotation shaft 221 in a plan view is used. In the example of fig. 13, the temperature adjustment unit 260 is provided in a belt-like range overlapping with the rotation orbit of the chamber 11 in a plan view. A plurality of temperature adjusting units 260 are provided in the circumferential direction to form temperature zones TZ set to different temperatures. In fig. 13, 3 temperature adjustment units 260 form 3 temperature zones TZ1 to TZ3 equally divided in a range of 120 degrees in the circumferential direction. The temperature zones TZ1 to TZ3 are set to 3 temperatures of low temperature, medium temperature, and high temperature in a range of, for example, 30 ℃ to 90 ℃. The chambers 11 for forming the emulsion 83 are sequentially arranged in the respective temperature zones TZ1 to TZ3 by rotating the cartridge 100, and thermal cycle is performed. The number of temperature adjustment units 260 (i.e., the number of temperature zones TZ) is not limited to this, and may be 4 or more. In the configuration of fig. 13, since the set temperature of each temperature adjustment unit 260 can be made constant, temperature control can be performed easily and with high accuracy.

In fig. 14, for example, a thermal cycle is performed by 1 temperature adjustment unit 260 in a plan view. In the example of fig. 14, the temperature adjustment unit 260 is provided at a position overlapping the rotation orbit of the chamber 11 in a plan view. The temperature adjustment unit 260 can accommodate 1 chamber 11 in the formed temperature zone TZ. The temperature adjusting unit 260 is formed of a Peltier element or the like to form a temperature zone TZ which can be arbitrarily changed in temperature within a variable range. In fig. 14, 1 temperature adjustment portion 260 forms a temperature zone TZ of which temperature changes in 3 stages. The temperature zone TZ is changed to 3-stage temperatures T1 to T3 of low temperature, medium temperature and high temperature in the range of 30 ℃ to 90 ℃, for example. The chamber 11 for forming the emulsion 83 is disposed at a position overlapping the temperature zone TZ by rotating the cartridge 100, and the temperature of the temperature adjusting section 260 is repeatedly changed to perform a thermal cycle. The number of stages of the temperature changed by the temperature adjusting portion 260 is not limited thereto, and may be 4 stages or more. In the configuration of fig. 14, 1 chamber 11 can be processed by 1 temperature controller 260, and thus the configuration of the apparatus for performing the thermal cycle process can be simplified. When a plurality of chambers 11 are provided, a plurality of temperature adjusting units 260 are provided, and the thermal cycle process can be performed simultaneously.

With such a configuration, the PCR process is performed in the emulsion 83. After the thermal cycle processing, the temperature adjustment unit 260 is released from the cassette 100. The cartridge 100 returns to room temperature.

"emulsion breaking

Returning to FIG. 11, the liquid reservoir 30 includes a 2 nd liquid reservoir 32 connected to the chamber 11 for storing a reagent 85 for demulsifying an emulsion by mixing. In step S15, after nucleic acids contained in the dispersoid are amplified, the reagent 85 for demulsifying the emulsion is transferred from the 2 nd liquid reservoir 32 to the chamber 11 in which the emulsion 83 is formed.

First, after the nucleic acid contained in the dispersoid is amplified, the closure 30a of the 2 nd liquid reservoir 32 is opened. Thus, the sealing member 30a prevents the reagent 85 for demulsifying the emulsion from being unintentionally conveyed from the 2 nd liquid storage portion 32. Therefore, it is possible to surely form the emulsion 83 and amplify the nucleic acid, and then demulsify the emulsion 83.

Next, by rotating the cartridge 100 about the rotation shaft 221, the reagent 85 for demulsifying the emulsion is transferred from the 2 nd liquid storage portion 32 to the chamber 11 for forming the emulsion 83. Thus, the nucleic acid amplified in the droplet 84 contained in the emulsion 83 can be taken out by the reagent 85 for demulsifying the emulsion. In this case, unlike the case of delivery by air pressure or the like, the reagent 85 for demulsifying the emulsion can be delivered to the chamber 11 only by rotating the cartridge 100 about the rotation shaft 221, and the emulsion 83 can be easily demulsified.

When a reagent 85 for demulsifying the emulsion is transferred into the chamber 11, the emulsion 83 is broken, and the magnetic particles PM containing the amplified nucleic acid are taken out from the droplets 84. The stirring may be performed in the chamber 11 by changing the rotation speed while rotating the cartridge 100. Thus, the reagent 85 and the emulsion 83 for demulsifying the emulsions by stirring can efficiently perform demulsification.

Cleaning for the 1 st time

The cartridge 100 contains a 2 nd chamber 12 connected to the chamber 11. The liquid reservoir 30 includes a 3 rd liquid reservoir 33 connected to the 2 nd chamber 12 for storing a reagent 86 for washing the detection object MD. In step S16, the reagent 86 for washing the test substance MD amplifies at least the nucleic acid contained in the dispersoid, and then the cartridge 100 is transferred from the 3 rd liquid reservoir 33 to the 2 nd chamber 12 by rotating the cartridge around the rotation shaft 221.

For example, after the nucleic acid contained in the dispersion is amplified, the closure 30a of the 3 rd liquid storage part 33 is unsealed at the same timing as unsealing the closure 30a of the 2 nd liquid storage part 32. Thus, the reagent 86 for washing the test substance MD can be prevented from being unintentionally conveyed from the 3 rd liquid reservoir 33 by the sealing member 30 a. When the 2 nd liquid storage portion 32 and the 3 rd liquid storage portion 33 are opened at the same timing, the operation of rotating the cartridge 100 for liquid feeding is completed 1 time.

As described above, the reagent 86 for washing the test substance MD contains alcohol. In step S14, the temperature of the cassette 100 is changed in the range of 30 ℃ to 90 ℃ when amplifying the nucleic acid contained in the dispersoid. Therefore, the reagent 86 for washing the test substance MD may be gasified by the influence of the temperature change. Thus, by transferring the reagent 86 for washing the test substance MD to the 2 nd chamber 12 after the nucleic acid amplification, the influence of the temperature change on the reagent 86 for washing the test substance MD can be suppressed. This constitution is particularly effective in that vaporization of the alcohol can be suppressed when the reagent 86 containing the substance to be detected MD is an alcohol.

In step S16, after transferring the reagent 85 for demulsifying the emulsion into the chamber 11, the nucleic acid bound to the magnetic particles PM in the demulsified liquid is transferred from the chamber 11 to the 2 nd chamber 12 by applying a magnetic force to the cartridge 100. Since the nucleic acid-supporting magnetic particles PM are taken out from the droplets 84 by demulsification, the magnetic particles PM move toward the 2 nd chamber 12 by the magnetic force and the rotation of the cartridge 100, as described above.

In the 2 nd chamber 12, the nucleic acid-supporting magnetic particles PM are washed with a reagent 86 for washing the detection object substance MD.

In step S16, the substance to be detected MD can be easily transferred to the 2 nd chamber 12 only by applying a magnetic force to the cartridge 100 from the outside without being transferred by air pressure or the like. Further, the substance MD to be detected in the liquid transfer chamber 11 is transferred by air pressure or the like, but the substance MD to be detected bonded only to the magnetic particles PM can be transferred to the 2 nd chamber 12 while retaining the liquid by transfer by magnetic force. As a result, the transfer of unnecessary components to the 2 nd chamber 12 is suppressed, and the nucleic acid in the 2 nd chamber 12 can be efficiently washed.

"variability" of the disease

The cartridge 100 contains a 3 rd chamber 13 connected to a 2 nd chamber 12. The liquid reservoir part 30 includes a 4 th liquid reservoir part 34 connected to the 3 rd chamber 13 and storing a reagent 87 for denaturing nucleic acid.

In step S17, the reagent 87 for denaturing nucleic acid is transferred from the 4 th liquid reservoir 34 to the 3 rd chamber 13 by rotating the cartridge 100 around the rotation shaft 221 after at least nucleic acid contained in the dispersoid is amplified. Thus, in step S17, in the cartridge 100, a process of denaturing nucleic acid can be performed also on nucleic acid after the process of washing nucleic acid in the 3 rd chamber 13. Further, even when the treatment for denaturing nucleic acid is performed, since the cassette 100 can be transported to the 3 rd chamber 13 only by rotating the cassette around the rotation shaft 221 without using a liquid transport such as air pressure, the treatment for denaturing nucleic acid can be performed easily.

The closure 30a of the 4 th liquid storage part 34 is opened at the same timing as the opening of the respective closures 30a of the 2 nd liquid storage part 32 and the 3 rd liquid storage part 33 after, for example, the nucleic acid contained in the dispersoid is amplified. Thus, the reagent 87 for denaturing nucleic acid can be prevented from being unintentionally delivered from the 4 th liquid reservoir part 34 by the sealing member 30 a. When the 2 nd, 3 rd and 4 th liquid storage portions 32, 33 and 34 are opened at the same timing, the operation of rotating the cartridge 100 for liquid feeding is completed 1 time.

In step S17, after the reagent 87 for denaturing nucleic acid is transferred to the chamber 11, the nucleic acid bound to the magnetic particles PM after the washing process is transferred from the 2 nd chamber 12 to the 3 rd chamber 13 by applying a magnetic force to the cartridge 100. As described above, the magnetic particles PM are moved toward the 3 rd chamber 13 by the magnetic force and the rotation of the cartridge 100.

In the 3 rd compartment 13, the reagent 87 for denaturing nucleic acid denatures the amplified double-stranded DNA into single strands.

"hybridization

The cartridge 100 contains a 4 th compartment 14 connected to a 3 rd compartment 13. The liquid storing part 30 includes a 5 th liquid storing part 35 connected to the 4 th chamber 14 for storing a reagent 88 containing a labeling substance LS that reacts with an amplification product of a nucleic acid.

In step S18, after the nucleic acid contained in the dispersoid is amplified by the reagent 88 containing the labeling substance LS, the cartridge 100 is transferred from the 5 th liquid reservoir 35 to the 4 th chamber 14 by rotating the cartridge around the rotation shaft 221.

The closure 30a of the 5 th liquid storage part 35 is opened at the same timing as the respective closures 30a of the 2 nd to 4 th liquid storage parts 32 to 34 are opened after, for example, the nucleic acid contained in the dispersoid is amplified. When the 2 nd to 5 th liquid storage portions 32 to 35 are opened at the same timing, the operation of rotating the cartridge 100 for liquid feeding is completed 1 time.

In step S18, after the reagent 88 containing the labeling substance LS is transferred to the 4 th chamber 14, the nucleic acid bound to the magnetic particles PM after the denaturation process is transferred from the 3 rd chamber 13 to the 4 th chamber 14 by applying a magnetic force to the cartridge 100. As described above, the magnetic particles PM are moved toward the 4 th chamber 14 by the magnetic force and the rotation of the cartridge 100.

In step S18, the nucleic acid transferred to the 4 th chamber 14 and the labeling substance LS are reacted by changing the temperature of the cassette 100. Thus, the cartridge 100 may be subjected to not only a process of denaturing nucleic acids but also a process of labeling for detecting nucleic acids as the detection target substance MD. Further, even when the labeling process of the nucleic acid is performed, since the cartridge 100 can be transported to the 4 th chamber 14 only by rotating the cartridge around the rotation shaft 221 without using a liquid transport such as air pressure, the labeling process of the nucleic acid can be easily performed.

The hybridization process may be performed by the temperature controller 260 (see fig. 13 and 14) that performs the thermal cycle process of the PCR. That is, the temperature adjusting section 260 raises the temperature of the cassette 100 at least by heating, thereby causing the nucleic acid in the 4 th chamber 14 to react with the labeling substance LS. Thus, only by heating at least the cartridge 100, both the process of amplifying the nucleic acid and the process of labeling the nucleic acid can be performed in the cartridge 100. Therefore, not only the preparation of the emulsion 83 but also the treatment of amplifying the nucleic acid and the treatment of labeling the nucleic acid using the cartridge 100 can be performed more easily.

In the 4 th chamber 14, the target DNA is labeled by binding of the labeling substance LS in the reagent 88 containing the labeling substance LS to the DNA as the detection target substance MD carried on the magnetic particles PM (see fig. 12).

2 nd washing

The cartridge 100 contains a 5 th compartment 15 connected to a 4 th compartment 14. The liquid reservoir section 30 includes a 6 th liquid reservoir section 36 connected to the 5 th chamber 15 for storing a reagent 89 for washing the detection object MD. In step S19, the reagent 89 for washing the test substance MD amplifies at least the nucleic acid contained in the dispersoid, and then the cartridge 100 is transferred from the 6 th liquid reservoir 36 to the 5 th chamber 15 by rotating the cartridge around the rotation shaft 221.

The closing body 30a of the 6 th liquid storage portion 36 is opened at the same timing as the opening of the closing bodies 30a of the 2 nd to 5 th liquid storage portions 32 to 35, for example. Thus, the operation of rotating the cartridge 100 for liquid feeding is completed 1 time.

In step S19, after the reagent 89 for washing the test substance MD is transferred to the 5 th chamber 15, the magnetic particles PM bound to the labeled nucleic acid are transferred from the 4 th chamber 14 to the 5 th chamber 15 by applying a magnetic force to the cartridge 100. As described above, the magnetic particles PM are moved toward the 5 th chamber 15 by the magnetic force and the rotation of the cartridge 100.

In the 5 th chamber 15, the magnetic particles PM bound to the labeled nucleic acid are washed by the reagent 89 for washing the detection object substance MD.

Detection

The cartridge 100 contains a 6 th chamber 16 connected to a 5 th chamber 15. The liquid reservoir 30 includes a 7 th liquid reservoir 37 connected to the 6 th chamber 16 for storing a reagent 90 for dispersing the detection target substance MD. Fig. 9 and 11 show an example in which 27 th liquid storage parts 37 are provided. Thus, a plurality of liquid storing parts for storing the same reagent may be provided. This makes it possible to easily secure a necessary liquid amount. The reagent 90 for dispersing the substance to be detected MD is, for example, PBS (phosphate buffered saline).

In step S20, the reagent 90 for dispersing the test substance MD amplifies at least the nucleic acid contained in the dispersoid, and then the cartridge 100 is transferred from the 7 th liquid reservoir 37 to the 6 th chamber 16 by rotating the cartridge around the rotation shaft 221.

The closure 30a of the 7 th liquid storage portion 37 is opened at the same timing as the closure 30a of the 2 nd to 6 th liquid storage portions 32 to 36 is opened, for example. Thus, the operation of rotating the cartridge 100 for liquid feeding is completed 1 time.

In the case of performing detection using a flow cytometer, for example, an operator punctures a pipette (not shown) inside the 6 th chamber 16 or opens a closure (not shown) for sample collection to collect a sample. The 6 th chamber 16 is filled with a predetermined amount of PBS, so that the amount of the sample liquid necessary for detecting the magnetic particles PM containing the labeled nucleic acid can be secured. The collected sample was tested for LS as a labeling substance by flow cytometry.

When detection is performed using an imaging unit, for example, the 6 th chamber 16 of the cartridge 100 is imaged, and the magnetic particles are counted by image processing. The 6 th chamber 16 is filled with a predetermined amount of PBS, and the magnetic particles PM containing the labeled nucleic acid are sufficiently dispersed in the 6 th chamber 16.

With the above-described configuration, in the sample processing method of embodiment 1, the processes shown in steps S13 to S19 or the processes shown in steps S13 to S20 of fig. 10 are performed using 1 cartridge 100.

(specific construction example of sample processing apparatus)

Next, referring to fig. 15 to 19, specific configuration examples of the sample processing apparatus 200 for performing the above-described sample processing method using the cartridge 100 will be shown. That is, the sample processing apparatus 200 can perform each of the processes of emulsion formation (step S13), emulsion PCR (step S14), emulsion disruption (step S15), 1 st washing (step S16), denaturation (step S17), hybridization (step S18), and 2 nd washing (step S19) on the nucleic acid (DNA) as the test substance MD. As will be described later, the sample processing apparatus 200 may execute the detection process (step S20).

As shown in fig. 15 and 16, the housing 201 of the sample processing apparatus 200 includes a main body 202 and a lid 203. The lid 203 is provided to cover substantially the entire upper surface of the main body 202. The installation section 210 for installing the cartridge 100 is provided on the upper surface of the main body 202. The lid 203 is vertically rotatable with respect to the main body 202, and openably and closably set in a state where the setting section 210 shown in fig. 15 is opened and a state where the setting section 210 shown in fig. 16 is covered. The main body 202 houses the rotating mechanism 220 and the control unit 230.

Internal structure of sample processing apparatus

As shown in fig. 17, the setting section 210 is configured as a support member that supports the cartridge 100 from below. The setting part 210 is provided at the upper end of the rotating shaft 221 of the rotating mechanism 220. The setting unit 210 is, for example, a turntable, and is fitted into the hole 51 of the cartridge 100 (see fig. 9) to align the center of the cartridge 100 with the position of the rotation shaft 221. The cover 203 is provided with a clamper 204. In a state where the gripper 204 closes the cover portion 203, the center portion of the upper surface of the cartridge 100 disposed on the setting portion 210 is rotatably supported.

In the example of fig. 17, the sample processing apparatus 200 includes a rotation mechanism 220, a transfer mechanism 240, and an unsealing mechanism 250.

The rotation mechanism 220 includes a rotation shaft 221 and a drive unit 222 including an electric motor. The rotation mechanism 220 drives the driving unit 222 to rotate the cartridge 100 set in the setting unit 210 about the rotation axis 221. The rotating mechanism 220 includes an encoder 223 for detecting the rotation angle of the driving unit 222 and an origin sensor 224 for detecting the origin position of the rotation angle. By driving the driving unit 222 based on the detected angle of the encoder 223 with reference to the position detected by the origin sensor 224, the cartridge 100 can be moved to an arbitrary rotational position.

The rotation mechanism 220 is configured to rotate the cartridge 100 about the rotation shaft 221 to perform at least a part of the sample processing. That is, the rotation mechanism 220 performs processes such as formation of an emulsion, transfer of a sample, transfer of a reagent to each of the chambers 11 to 16 (see fig. 11), stirring of the reagent and the sample, and transfer of the magnetic particles PM between the chambers 11 to 16 in the circumferential direction in the cartridge 100 by rotation.

The transfer mechanism 240 includes a magnet 241, and has a function of moving the magnetic particles PM in the cartridge 100 in the radial direction by a magnet moving mechanism 242. The transfer mechanism 240 is disposed below the installation portion 210. The magnet moving mechanism 242 is constituted by a combination of linear motion mechanisms, and is constituted so as to move the magnet 241 in the radial direction. The magnet moving mechanism 242 is configured to move the magnet 241 in a direction toward or away from the cartridge 100. By bringing the magnets 241 close to each other, the magnetic particles PM in the cartridge 100 are collected and by separating the magnets 241, the magnetic collection of the magnetic particles PM is released. The transfer mechanism 240 moves the magnet 241 in the radial direction in a magnetic-flux-collected state, thereby moving the magnetic particles PM (see fig. 11) in the cartridge 100 to the inside and outside of the chamber. Thereby, the transfer mechanism 240 transfers the magnetic particles PM to the 2 nd chamber 12 connected to the chamber 11. Similarly, the transfer mechanism 240 transfers the magnetic particles PM to another chamber.

The unsealing mechanism 250 includes a needle member 251 that can advance and retreat from above the cartridge 100 disposed in the installation section 210 toward the cartridge 100. The unsealing mechanism 250 advances and retracts the needle member 251 by a drive source such as a solenoid or a motor. When the rotating mechanism 220 disposes the sealing body 30a (see fig. 9) at a position directly below the needle member 251, the unsealing mechanism 250 causes the needle member 251 to protrude and abut against the cartridge 100, and unseals the sealing body 30a in the cartridge 100 by pressing. After the opening, the unsealing mechanism 250 moves the needle member 251 to the retreat position separated from the cartridge 100 and brought into non-contact.

The sample processing apparatus 200 further includes a temperature adjustment unit 260 that changes the temperature of the cartridge 100 to cause the reaction between the substance MD to be detected contained in the dispersoid in the emulsion 83 and the reagent 81. Thus, the substance MD and the reagent 81 can be reliably reacted by a temperature change in a state where the substance MD and the reagent 81 for processing the substance MD are accommodated in the droplets 84 (see fig. 12) of the dispersoid and partitioned. In addition, the substance MD and the reagent 81 can be efficiently reacted by a temperature change in the droplets 84 contained in the emulsion 83, as compared with the case where the reaction between the substance MD and the reagent 81 is naturally performed at the temperature of the installation environment of the cartridge 100.

The temperature adjustment unit 260 is disposed at a position just above the cartridge 100 disposed in the setting unit 210. The temperature adjustment portion 260 is disposed so as to face the cartridge 100 on the inner surface of the cover portion 203. The shape of the plane of the temperature adjustment portion 260 may adopt the configuration shown in fig. 13 or 14. The temperature adjusting section 260 is constituted by a heater or a Peltier element.

The one or more temperature adjusting units 260 amplify the nucleic acid contained in the dispersoid by periodically changing the temperature of the cartridge 100 over a plurality of temperature ranges. Thus, the droplets 84 contained in the emulsion 83 can be subjected to a so-called thermal cycle treatment to efficiently amplify the nucleic acid.

The temperature adjusting unit 260 is configured to be movable relatively to a position away from a position in contact with the cartridge 100 disposed in the installation unit 210. In the example of fig. 17, the temperature adjustment unit 260 is movable by the movement mechanism 261 to a position in contact with the surface of the cartridge 100 and a position away from the surface of the cartridge 100. The moving mechanism 261 includes a driving source such as a solenoid or a motor. The temperature adjusting part 260 changes the temperature of the cartridge 100 in a state of being in contact with the cartridge 100. Thus, when the cartridge 100 is heated in a non-contact manner, the temperature adjustment unit 260 is brought into contact with the cartridge 100, so that heat can be efficiently transferred, and the temperature of the cartridge 100 can be easily changed.

The temperature adjustment unit 260 may be fixedly provided, and the installation unit 210 may be configured to move the cartridge 100 in a direction toward or away from the temperature adjustment unit 260. The sample processing apparatus 200 is further provided with a temperature sensor 262. The temperature sensor 262 detects the temperature of the cartridge 100 by, for example, infrared rays. The temperature adjusting section 260 is controlled based on the detection result of the temperature sensor 262.

The sample processing device 200 may also include a detection unit 270 that detects a signal based on the labeling substance LS. As described above, the detection unit 270 may be, for example, a flow cytometer, an imaging unit (imaging unit), or the like. In fig. 17, an example in which the detection unit 270 is configured by an imaging unit is shown. The detection unit 270 is disposed at a position facing the cartridge 100 disposed in the installation unit 210 through an opening formed in the upper surface of the main body 202. Thereby, the detection unit 270 images the inside of the 6 th chamber 16 (see fig. 11). By performing image processing on the captured image, each magnetic particle PM in the 6 th chamber 16 can be recognized, and a fluorescent color due to the labeling substance LS bonded to the magnetic particle PM can be obtained.

The sample processing apparatus 200 may not include the detection unit 270. At this time, as shown in fig. 19, the processed cartridge 100 is taken out of the sample processing apparatus 200 and subjected to detection processing using another measuring apparatus.

Fig. 18 shows a configuration of control of the sample processing apparatus 200.

The sample processing apparatus 200 includes a control unit 230. The control section 230 includes, for example, a processor and a memory. The processor is configured by, for example, a CPU, MPU, FPGA, or the like. The memory is constituted by, for example, ROM, RAM, and the like. The control unit 230 receives signals from the respective units of the sample processing apparatus 200 and controls the respective units of the sample processing apparatus 200.

The sample processing apparatus 200 includes a storage unit 231. The storage unit 231 stores a program or the like for causing the processor to function as the control unit 230 of the sample processing apparatus 200. The storage unit 231 is configured by, for example, a flash memory, a hard disk, or the like.

The sample processing device 200 includes a communication unit 232. The communication unit 232 can transmit and receive information to and from the external device. The communication unit 232 includes, for example, a communication module and an interface for external connection. The communication unit 232 is capable of performing communication with a terminal 600 (see fig. 19) capable of communicating with the sample processing device 200 and communication with a server 650 (see fig. 19) via a network by wired or wireless communication. The sample processing apparatus 200 is configured to be capable of performing various operations and displaying various information by using the external terminal 600. The terminal 600 includes, for example, a tablet type terminal, a mobile information terminal such as a smart phone, and an information terminal such as a PC (personal computer).

The control unit 230 controls each unit of the sample processing apparatus 200 to execute the sample processing method according to embodiment 1.

Operation of sample processing apparatus

Next, the processing operation of the sample processing apparatus 200 will be described with reference to fig. 9 to 17. The processing operation of the sample processing apparatus 200 is controlled by the control unit 230. As a preparation operation, a dispersion including the sample 80 and the reagent 81 after the steps S11 and S12 in fig. 10 is injected into the cartridge 100 by an operator. Further, the cartridge 100 is installed in the installation portion 210 (see fig. 15) of the sample processing device 200, and the lid portion 203 (see fig. 16) is closed. In this state, the processing operation is started.

In step S13, the controller 230 controls the rotating mechanism 220 so as to transfer the dispersoid contained in the sample containing portion 38 of the cartridge 100 to the chamber 11 by rotation. The control unit 230 controls the unsealing mechanism 250 to unseal the closure 30a of the 1 st liquid storage portion 31. After the unsealing, the controller 230 controls the rotating mechanism 220 so as to transfer the dispersant 82 stored in the 1 st liquid storage unit 31 provided in the cartridge 100 to the chamber 11 by rotation. The dispersoid and the dispersant 82 are transferred into the chamber 11 by centrifugal force.

Thus, not only the generation of the emulsion 83 but also the operation of storing the dispersion medium 82 in the chamber 11 can be performed only by rotating the cartridge 100 by the rotating mechanism 220. Therefore, for example, since air pressure is not required to be added to the 1 st liquid storage part 31, the sample can be processed more easily. Further, since the dispersing agent 82 is transferred from the liquid storage section 30 to the chamber 11, it is necessary to provide a mechanism without air pressure, and the like, and the device configuration can be further simplified.

The controller 230 controls the rotation mechanism 220 to repeat the operation of changing the rotation speed of the cartridge 100, thereby forming an emulsion 83 in which a dispersoid including the sample 80 and the reagent 81 is dispersed in the dispersing agent 82 in the chamber 11.

For example, the controller 230 controls the rotating mechanism 220 so as to repeat the operation of reversing the rotation direction of the cartridge 100 in a cycle of 165 milliseconds to 330 milliseconds when the emulsion 83 is formed. Thus, only by repeating the operation of reversing the rotation direction at a cycle of 165 milliseconds to 330 milliseconds based on the results of the experiment described later, the test substance MD can be processed to produce the emulsion 83 suitable for detection.

The control unit 230 forms an emulsion 83 containing droplets 84 of a dispersoid containing 1 molecule or 1 detection target substance MD in the chamber 11 by the rotation of the cartridge 100. This makes it possible to separate each of 1 test substance MD contained in the sample into 1 droplet 84 contained in the emulsion 83 and to reliably treat the substance MD. As a result, since the sample can be processed with high accuracy even if the sample contains only a very small amount of the analyte MD, the detection accuracy can be improved when the processed analyte MD is detected.

In step S14, the control unit 230 controls the temperature adjustment unit 260 so that the temperature of the cartridge 100 is changed in the range of 30 ℃ to 90 ℃. Thus, the emulsion PCR treatment was performed. For example, in the configuration example of fig. 13, control unit 230 controls 3 temperature adjustment units 260 to form different temperature zones TZ1 to TZ 3. Further, the rotation mechanism 220 is controlled to sequentially move the chamber 11 to the temperature zones TZ1 to TZ 3. In each temperature zone TZ, the controller 230 controls the movement mechanism 261 so that the temperature adjuster 260 and the cartridge 100 are in contact with each other, and controls the inside of the chamber 11 to a desired temperature.

The control unit 230 controls the unsealing mechanism 250 to unseal the sealing member 30a of the 2 nd liquid storage portion 32 after the emulsion 83 is formed in the chamber 11 and the substance to be detected MD and the reagent 81 are reacted by changing the temperature of the cartridge 100. Thus, the sealing member 30a prevents the reagent 85 for demulsifying the emulsion from being unintentionally conveyed from the 2 nd liquid storage portion 32. Therefore, by providing the unsealing mechanism 250 for unsealing the closed body 30a, it is possible to surely form the emulsion 83 and amplify the nucleic acid, and then to perform demulsification of the emulsion 83. Similarly, the control unit 230 controls the unsealing mechanism 250 to unseal the respective closures 30a of the 3 rd to 7 th liquid storage portions 33 to 37.

In step S15, the controller 230 controls the rotating mechanism 220 so that the reagent 85 for demulsifying the emulsion is transferred from the 2 nd liquid storage unit 32 provided in the cartridge 100 to the chamber 11 for forming the emulsion 83 by the rotation. The emulsion 83 in the chamber 11 is broken by the reagent 85 for breaking the emulsion. Thus, not only the process of forming the emulsion 83 but also the process of demulsifying the emulsion 83 may be performed in the cartridge 100 by the sample processing apparatus 200. In this case, since the reagent 85 for demulsifying the emulsion is supplied to the chamber 11, a mechanism for adding air pressure is not necessary, and the apparatus configuration can be further simplified.

At this time, when the closing bodies 30a of the 3 rd to 7 th liquid storing parts 33 to 37 are also opened, the reagents stored in these liquid storing parts 30 can be transported to the corresponding chambers by 1 liquid transport operation.

In this way, since the control unit 230 controls the unsealing mechanism 250 so as to unseal the sealing body 30a of the 3 rd liquid containing portion 33 containing the reagent 86 for washing the substance MD after the reaction of the substance MD and the reagent 81 is performed at least by the temperature adjusting unit 260, the reagent 86 for washing the substance MD is transported to the 2 nd chamber 12 after being amplified by the temperature change by the temperature adjusting unit 260. This can suppress the influence of temperature change on the reagent 86 for washing the test substance MD.

In step S16, the control unit 230 controls the transfer mechanism 240 so that the detection target substance MD is transferred from the chamber 11 to the 2 nd chamber 12 after the emulsion 83 is demulsified. That is, the controller 230 moves the magnetic particles PM in the radial direction by the magnetic force of the transfer mechanism 240, and moves the cartridge 100 in the circumferential direction with respect to the magnetic particles PM by the rotation of the rotation mechanism 220. The magnetic particles PM bound to the detection target substance MD move to the 2 nd chamber 12 through the passage 40 by a combination of the magnetic force and the rotation of the cartridge 100. The control unit 230 washes the magnetic particles PM with the reagent 86 for washing the detection object MD. Thus, the nucleic acid as the detection target substance MD can be amplified in the chamber 11, and after the nucleic acid is taken out from the droplet 84 by demulsification, the nucleic acid can be washed in the 2 nd chamber 12.

Further, the control unit 230 unseals the closure 30a of the liquid storage portion 30 which stores the reagent 87 for denaturing the nucleic acid after the reaction of the substance to be detected MD and the reagent 81 by the temperature adjusting unit 260 (after step S14). Therefore, the reagent 87 for denaturing the nucleic acid is transferred to the 3 rd chamber 13 after the amplification is performed by the temperature change using the temperature adjustment unit 260. This can suppress the influence of temperature change on the reagent 87 for denaturing nucleic acid.

In step S17, the control unit 230 controls the transfer mechanism 240 to transfer the detection target substance MD stored in the 2 nd chamber 12 to the 3 rd chamber 13. In the 3 rd chamber 13, the reagent 87 for denaturing nucleic acid is transferred from the 4 th liquid reservoir 34 by rotation. As a result, in the 3 rd chamber 13, the nucleic acid (double-stranded DNA) bound to the magnetic particles PM is denatured into single strands. Thus, in the cartridge 100, the nucleic acid after the treatment of washing the nucleic acid in the 2 nd chamber 12 can be further subjected to a treatment of denaturing the nucleic acid. Further, even when the treatment for denaturing nucleic acid is performed, since the cassette 100 can be transported to the 3 rd chamber 13 only by rotating the cassette around the rotation shaft 221 without using a liquid transport such as air pressure, the treatment for denaturing nucleic acid can be performed with a simple apparatus configuration.

Further, the control unit 230 unseals the closure body 30a of the liquid storage portion 30 which stores the reagent 87 for denaturing the nucleic acid after the reaction of the substance to be detected MD and the reagent 81 by the temperature adjusting unit 260 (after step S14). Therefore, the reagent 87 for denaturing the nucleic acid is transferred to the 3 rd chamber 13 after the amplification is performed by the temperature change using the temperature adjustment unit 260. This can suppress the influence of temperature change on the reagent 87 for denaturing nucleic acid.

In step S18, the control unit 230 controls the transfer mechanism 240 to transfer the detection target substance MD stored in the 3 rd chamber 13 to the 4 th chamber 14. In the 4 th chamber 14, the reagent 88 containing the labeling substance LS that reacts with the amplification product of nucleic acid is transferred from the 5 th liquid reservoir 35 by rotation. Thus, the cartridge 100 may be subjected to not only a process of denaturing nucleic acids but also a process of labeling for detecting nucleic acids as the detection target substance MD. Further, even when the process of labeling the nucleic acid is performed, since the cartridge 100 can be transported to the 4 th chamber 14 only by rotating the cartridge by the rotating mechanism 220 without using a liquid transport such as air pressure, the process of labeling the nucleic acid can be performed by a simple apparatus configuration.

The controller 230 controls the temperature adjuster 260 to increase the temperature of the cassette 100, thereby reacting the nucleic acid in the 4 th chamber 14 with the label LS. As a result, the control unit 230 performs labeling processing of the amplified product of the nucleic acid in the 4 th chamber 14. Thus, both the process of amplifying nucleic acid and the process of labeling nucleic acid can be performed in the cartridge 100 by the common temperature adjustment unit 260. Therefore, not only the preparation of the emulsion 83 but also the treatment of amplifying the nucleic acid and the treatment of labeling the nucleic acid using the cartridge 100 can be performed more simply by a simple apparatus configuration.

In step S19, the control unit 230 controls the transfer mechanism 240 to transfer the detection target substance MD stored in the 4 th chamber 14 to the 5 th chamber 15. In the 5 th chamber 15, the reagent 89 for washing the detection target substance MD is transferred from the 6 th liquid reservoir 36 by rotation. As a result, the control unit 230 cleans the magnetic particles PM after the marking process in the 5 th chamber 15.

In step S20, the control unit 230 controls the transfer mechanism 240 to transfer the detection target substance MD stored in the 5 th chamber 15 to the 6 th chamber 16. In the 6 th chamber 16, the reagent 90 (PBS) for dispersing the analyte MD is transferred from the 7 th liquid reservoir 37 by rotation. As a result, the controller 230 disperses the magnetic particles PM subjected to the labeling process in the reagent in the 6 th chamber 16.

The controller 230 controls the rotation mechanism 220 to dispose the 6 th chamber 16 in the imaging field of view of the detector 270. The control unit 230 acquires an image of the 6 th chamber 16 from the detection unit 270. The control unit 230 counts the number of each magnetic particle PM based on the acquired image, and acquires the fluorescence color and fluorescence intensity of each magnetic particle PM. The controller 230 creates a scattergram of normal DNA and variant DNA from the acquired fluorescence color and fluorescence intensity of the magnetic particles PM, for example.

If the sample processing device 200 does not include the detection unit 270, the control unit 230 ends the process without executing step S20. At this time, as shown in fig. 19, the operator takes the processed cartridge 100 out of the sample processing apparatus 200 and performs detection using another measuring apparatus.