JP2019190991A - Microparticle measurement device and microparticle measurement method - Google Patents

Abstract

To provide a technique that can improve the measurement accuracy.SOLUTION: Provided is a microparticle measurement device or the like that comprises at least: a sheath liquid introduction unit that introduces a sheath liquid from a sheath liquid storage unit to a flow path in which a sample liquid including microparticles mixes with the sheath liquid; a light emission unit that emits light toward the microparticles passing through the flow path; and a detection unit that detects light from the microparticles. The sheath liquid introduction unit comprises a temperature control unit that controls the temperature of the sheath liquid.SELECTED DRAWING: Figure 1

Description

  The present technology relates to a microparticle measurement apparatus and a microparticle measurement method.

  Currently, a technique called flow cytometry is used to analyze microparticles such as cells and microorganisms. This flow cytometry irradiates light to the microparticles that flow so as to be contained in the sheath flow sent into the flow path, and detects the fluorescence and scattered light emitted from the individual microparticles. This is an analysis method that performs analysis and the like. An apparatus used for this flow cytometry is called a flow cytometer.

  In a flow cytometer, it is necessary to align microparticles to be analyzed in a line in a flow path. More specifically, the sheath flow is caused to flow into the flow path at a constant flow rate, and in this state, the sample flow containing microparticles is also slowly injected into the flow path. At this time, according to the principle of Laminar Flow, the respective flows are not mixed with each other, and a laminar flow (laminar flow) is formed. Then, the inflow amounts of the sheath flow and the sample flow are adjusted according to the size of the microparticles to be analyzed, and the microparticles are allowed to flow in an aligned state.

  When analyzing fine particles contained in a laminar flow, it is very important to control the flow rate in order to improve measurement accuracy. Here, in Patent Document 1, a flow rate of the sheath liquid is set by providing a pressure sensor in the flow path of the sheath liquid and changing the pressure value applied to the sheath liquid based on the water pressure sensed by the pressure sensor. Techniques for controlling are disclosed.

JP-A 64-88251

  However, in the analysis of microparticles contained in the laminar flow, development of a further technique that can stabilize the flow velocity and improve the measurement accuracy is desired.

  Thus, the main object of the present technology is to provide a technology capable of improving the measurement accuracy.

In the present technology, first, a sheath liquid introduction part that introduces a sheath liquid from a sheath liquid storage part and a flow path through the flow path where the sample liquid containing microparticles and the sheath liquid join each other. A light irradiation unit for irradiating light to the microparticles, and a detection unit for detecting light from the microparticles, and the sheath liquid introduction unit includes a temperature control unit for controlling the temperature of the sheath liquid; A fine particle measuring apparatus is provided.
In the present technology, the sheath liquid introduction section connects the sheath liquid storage section and the flow path via a tube, and the tube may include at least one temperature sensor. .
In this case, the temperature sensor may be provided in contact with the surface of the tube. The temperature sensor may be provided in the vicinity of the flow path. Furthermore, the temperature sensor may be connected to the temperature control unit through a circuit. In addition, the sheath liquid introduction unit may further include a temperature adjustment unit that adjusts the temperature of the sheath liquid.
In this case, the temperature control unit may include a temperature control element, a metal member, and a temperature inspection unit that inspects the temperature of the metal member. The metal member may be provided so as to sandwich the tube. Furthermore, the temperature control element and the temperature inspection unit may be connected to the temperature control unit through a circuit. In addition, the temperature control unit may control the temperature control element by PI control. Further, the temperature sensor may be provided between the sheath liquid storage unit and the temperature control unit.
Moreover, in this technique, the said light irradiation part may irradiate light to the microparticle which flows through the said flow path from different axes.
Furthermore, in this technique, you may further provide the fractionation part which fractions the target microparticle.
In this case, the flow path may be formed in a microchip, and the sorting by the sorting unit may be performed outside the microchip. Alternatively, the flow path is formed in the microchip and the sorting is performed. Sorting by the collecting unit may be performed in the microchip.

  Further, according to the present technology, a sheath liquid introduction step of introducing the sheath liquid from the sheath liquid storage unit to the flow path where the sample liquid containing the microparticles and the sheath liquid are joined, and the flow path is passed. There is also a microparticle measurement method in which at least a light irradiation process for irradiating light to microparticles and a detection process for detecting light from the microparticles are performed, and the temperature of the sheath liquid is controlled in the sheath liquid introduction process. provide.

  In the present technology, “microparticles” may include a wide range of living body-related microparticles such as cells, microorganisms, and liposomes, or synthetic particles such as latex particles, gel particles, and industrial particles.

  Biologically relevant microparticles include chromosomes, liposomes, mitochondria, organelles (organelles) that constitute various cells. The cells include animal cells (for example, blood cells) and plant cells. Microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, biologically relevant microparticles include biologically relevant polymers such as nucleic acids, proteins, and complexes thereof. The industrial particles may be, for example, an organic or inorganic polymer material, a metal, or the like. Organic polymer materials include polystyrene, styrene / divinylbenzene, polymethyl methacrylate, and the like. Inorganic polymer materials include glass, silica, magnetic materials, and the like. Metals include gold colloid, aluminum and the like. The shape of these fine particles is generally spherical, but in the present technology, it may be non-spherical, and the size, mass and the like are not particularly limited.

According to the present technology, it is possible to improve measurement accuracy.
Note that the effects described here are not necessarily limited, and may be any of the effects described in the present disclosure.

It is a schematic conceptual diagram which shows 1st embodiment of the microparticle measuring device which concerns on this technique. It is a schematic conceptual diagram which shows 2nd embodiment of the microparticle measuring device which concerns on this technique. It is a perspective view which shows an example of embodiment of a temperature control unit. It is an exploded view of the temperature control unit shown in FIG. A and B are schematic views showing an example of a configuration of a microchip M1 that can be used in the fine particle measuring apparatus of FIG. 1 or 2. FIG. FIGS. 4A to 4C are schematic diagrams illustrating an example of the configuration of the orifice M11 of the microchip M1 that can be used in the microparticle measurement apparatus of FIG. It is a schematic conceptual diagram which shows 3rd embodiment of the microparticle measuring device which concerns on this technique. It is a schematic diagram which shows an example of a structure of the microchip M2 which can be used for the microparticle measuring apparatus of FIG. It is a perspective view which shows an example of a structure of the microchip M2 which can be used for the microparticle measuring apparatus of FIG. It is a cross-sectional schematic diagram corresponding to the QQ cross section in FIG.

Hereinafter, preferred embodiments for carrying out the present technology will be described with reference to the drawings.
The embodiment described below shows an example of a typical embodiment of the present technology, and the scope of the present technology is not interpreted narrowly. The description will be given in the following order.
1. Fine particle measuring apparatus 100
(1) Sheath liquid introduction part 1
(2) Light irradiation unit 2
(3) Detection unit 3
(4) Sorting unit 4
(4-1) Microchip M1
(4-2) Microchip M2
(5) Analysis unit 5
(6) Storage unit 6
(7) Display unit 7
(8) Input unit 8
(9) Control unit 9
(10) Insertion section 101
(11) Sample feeding part 102
(12) Drainage unit 103
2. Microparticle measurement method (1) Sheath liquid introduction process (2) Light irradiation process (3) Detection process

1. Fine particle measuring apparatus 100
FIG. 1 is a schematic conceptual diagram illustrating a first embodiment of a microparticle measurement apparatus according to the present technology. The microparticle measurement apparatus 100 according to the present technology includes at least a sheath liquid introduction unit 1, a light irradiation unit 2, and a detection unit 3. In addition, as necessary, it includes a sorting unit 4, an analysis unit 5, a storage unit 6, a display unit 7, an input unit 8, a control unit 9, an insertion unit 101, a sample liquid feeding unit 102, a drainage unit 103, and the like. May be. Hereinafter, each part will be described in detail.

(1) Sheath liquid introduction part 1
The sheath liquid introduction unit 1 introduces the sheath liquid from the sheath liquid storage unit 110 to the flow path where the sample liquid containing microparticles and the sheath liquid merge. Further, the sheath liquid introduction unit 1 includes a temperature control unit 11 that controls the temperature of the sheath liquid.

  Conventionally, the change in the environmental temperature in the test chamber has an influence on the viscosity of the sheath liquid, so that the flow rate of the sheath liquid changes and adversely affects the measurement result. In contrast, in the present technology, by controlling the temperature of the sheath liquid, it is possible to remove the influence due to the change in the environmental temperature and improve the measurement accuracy.

  Further, by controlling the temperature of the sheath liquid, the pain of the sample can be prevented and the viability of the sample can be maintained. Furthermore, in the case where droplets are formed when the target microparticles are sorted by the sorting unit 4 described later, as in the microparticle measuring apparatus 100 shown in FIG. 1 or 2, the temperature of the sheath liquid is controlled. By making the flow rate of the sheath liquid flowing into the flow path constant, droplet formation can be stabilized and measurement accuracy can be improved.

  In the present technology, the temperature controlled by the temperature control unit 11 is not particularly limited, and can be controlled by appropriately setting a target temperature.

  In the present technology, the sheath liquid introduction unit 1 connects the sheath liquid storage unit 110 and the flow path via a tube, and the tube includes at least one temperature sensor. preferable.

  The method for installing the temperature sensor is not particularly limited, but the temperature sensor is preferably provided so as to be in contact with the surface of the tube. If it is attempted to directly detect the temperature of the sheath liquid by installing a temperature sensor so as to be inserted into the tube, contamination risk or the like occurs, and the quality of the sheath liquid is affected. Therefore, by providing the tube so as to be in contact with the surface of the tube, it is possible to indirectly measure the temperature of the sheath liquid and avoid this risk.

  The temperature sensor is installed in contact with the surface of the tube by a fixing mechanism using a heat insulating material or the like. By using a heat insulating material, heat detection loss can be reduced as much as possible.

  Moreover, although the installation position of a temperature sensor is not specifically limited, It is preferable that it is provided in the said flow path vicinity. Thereby, the temperature of the sheath liquid near the inlet of the flow path can be controlled to be constant, and the measurement accuracy can be improved.

  Furthermore, as shown in FIG. 2, the temperature sensor is preferably provided between the sheath liquid storage unit 110 and a temperature adjustment unit 12 described later. There is a difference between the temperature of the sheath liquid passing through the tube and the temperature of the tube surface through which the sheath liquid passes depending on the environmental temperature, and it is not constant. In order to set the sheath liquid temperature as the target temperature, it is necessary to correct the target value of the tube surface temperature using the environmental temperature. Therefore, by providing a temperature sensor between the sheath liquid storage unit 110 and the temperature control unit 12 and measuring the environmental temperature, it is possible to perform correction based on the environmental temperature via the temperature control unit 11, and more accurately. In addition, the temperature of the sheath liquid can be controlled.

  In the present technology, the temperature sensor is preferably connected to the temperature control unit 11 through a circuit. With this circuit system, the temperature of the sheath liquid can be more accurately stabilized.

  When there are a plurality of temperature sensors, it is preferable that each temperature sensor is connected to the temperature control unit 11 through a circuit. In this case, in the temperature control unit 11, for example, from the value obtained from the temperature sensor 111b provided between the sheath liquid storage unit 110 and the temperature adjustment unit 12, and from the temperature sensor 111a provided in the vicinity of the flow path. A correlation between the two temperatures of the obtained values and the temperature of the sheath fluid that actually passes through the tube is obtained by experiment, and is set in the control program of the temperature adjustment unit 11 as a correction value table. . Then, by using this table, the target temperature that the surface of the tube should reach is corrected according to the environmental temperature, thereby removing the influence of the temperature difference between the inside and outside of the tube and bringing the temperature of the sheath fluid flowing in the tube closer to the target temperature. Control becomes possible.

  In the present technology, it is preferable that the sheath liquid introduction unit 1 further includes a temperature adjustment unit 12 that adjusts the temperature of the sheath liquid.

  FIG. 3 is a perspective view showing an example of an embodiment of the temperature control unit 12, and FIG. 4 is an exploded view of the temperature control unit 12 shown in FIG. The configuration of the temperature control unit 12 includes, for example, a temperature control element 121, a metal member 122, and a temperature inspection unit 123. The A side of the tube shown in FIG. 4 is connected to the sheath liquid reservoir 110 and is the side into which the sheath liquid flows, and the B side of the tube shown in FIG. 4 is the side from which the sheath liquid is discharged into the flow path. .

  The temperature control element 121 is, for example, a semiconductor cooling / heating element that generates a temperature difference by flowing a direct current, and specifically includes, for example, a Peltier element.

  Although the member which comprises the metal member 122 will not be specifically limited if it is a metal, For example, they are copper, brass, etc. The metal member 122 is preferably provided so as to sandwich the tube. As a result, the temperature of the sheath liquid flowing in the tube can be adjusted more accurately.

  In the present technology, for example, as illustrated in FIG. 3, by installing the temperature control element 121 so as to be in contact with the metal member 122, the metal member 122 itself can be heated or cooled. The temperature of the sheath liquid passing through the tube can be indirectly controlled.

  The temperature inspection unit 123 inspects the temperature of the metal member 122. Specifically, for example, a temperature sensor or the like. For example, as shown in FIG. 4, the temperature inspection unit 123 is attached to the metal member 122 and controls the temperature of the metal member 122. As a result, if a sudden failure occurs and the temperature of the metal member 122 shows an abnormal value, an error message is transmitted to the temperature control unit 11 based on the value based on the temperature sensor to avoid a dangerous situation. Can be controlled.

  Further, as shown in FIG. 4, the temperature control unit 12 may be provided with a heat sink 124 and a fan 125, and by providing these, the temperature control element 121 can be radiated and cooled. Moreover, it can be set as the structure which can be self-contained as the temperature control unit 12, and usability improves.

  It is preferable that the temperature control element 121 and the temperature inspection unit 123 are connected to the temperature control unit 11 through a circuit. With this circuit system, the temperature control in the temperature control element 121 and the temperature inspection unit 123 can be efficiently performed. Specifically, for example, the temperature control unit 11 sets a target temperature of the metal member 122 and controls the temperature control element 121 so that the value obtained from the temperature inspection unit 123 matches the target temperature.

  In this case, the temperature control unit 11 can control the temperature control element 121 by PI control (feedback control using a proportional term and an integral term).

  In the present technology, in the control by the PI control, it is possible to set a condition such that the integral term is not accumulated until the proportional term becomes small to some extent (until the current temperature of the temperature inspection unit 123 approaches the target temperature). As a result, even in a system that takes time for heating and cooling, it is possible to reduce the occurrence of overshoot in which the integral term during that time becomes an excessive value and exceeds the target value.

  Furthermore, the change value of the integral term near the target temperature can be intentionally reduced. As a result, in the control system having a long feedback time, it is possible to prevent the temperature fluctuation from excessively oscillating across the target temperature. That is, the control system is such that it reaches the vicinity of the target temperature as soon as possible, but it takes time to reach the target temperature from there.

  In addition, parameters for use in PI control may be prepared for heating and cooling, and may be switched according to operating conditions. This is because the characteristics are greatly different between the heating operation and the cooling operation of the temperature control element 121, so that control according to each characteristic is performed.

  In the present technology, it is more preferable that a temperature sensor 111a is provided in the vicinity of the flow path, and the temperature sensor 111a, the temperature control element 121, and the temperature inspection unit 123 are connected to the temperature control unit 11 through a circuit. Thereby, for example, the temperature control unit 11 calculates the difference between the temperature detected by the temperature sensor near the flow path and the target temperature to be reached there. And the system which adds to the current temperature of the temperature test | inspection part 123 and corrects it as the target temperature of the new temperature test | inspection part 123 is employ | adopted there, and the temperature of the sheath liquid in a tube is stabilized more efficiently. Can be made.

  Specifically, for example, when the temperature detected by the temperature sensor 111a in the vicinity of the flow path is 2 ° C. lower than the target temperature, a temperature obtained by adding 2 ° C. to the current temperature of the temperature inspection unit 123 is Control to reset the target temperature.

By performing this control periodically, the target temperature of the temperature inspection unit 123 is changed as needed until there is no difference between the temperature detected by the temperature sensor 111a near the flow path and the final target temperature. Finally, the temperature detected by the temperature sensor 111a in the vicinity of the flow path can be quickly converged to the target temperature.

  This also causes a problem that the temperature of the sheath liquid is not constant because the sheath liquid radiates heat in the course of reaching the flow path after being heated and cooled by the temperature control unit 12, and the temperature control unit 12 Due to the loss of time such as the conduction time of heating and cooling, the movement time of the sheath liquid, and the conduction time from the inside of the tube in the vicinity of the flow path to the outer skin, the temperature of the sheath liquid as a result of control from the driving to heat and cool the sheath liquid It is possible to solve the problem that it takes a long time to detect in the vicinity of the flow path. That is, it is possible to provide a feedback system that comprehensively corrects variation factors such as variations in heat dissipation and heating / cooling performance of equipment and delay time effects such as movement of heat transfer liquid.

  In the present technology, temperature sensors are provided in the vicinity of the flow path and between the sheath liquid storage unit and the temperature adjustment unit, and the temperature sensors 111a and 111b, the temperature control element 121, and the temperature inspection unit are provided. 123 is particularly preferably connected to the temperature control unit 11 through a circuit. Thus, in addition to the above-described control, as described above, the influence of the temperature difference between the inside and outside of the tube is removed based on the value obtained from the temperature sensor 111b provided between the temperature control unit 12 and the inside of the tube flows. It is possible to control the sheath liquid temperature closer to the target temperature.

(2) Light irradiation unit 2
The light irradiation part 2 irradiates light to the microparticle used as a measuring object which flows through the said flow path.

  Although the kind of light irradiated from the light irradiation part 2 is not specifically limited, In order to generate | occur | produce fluorescence and scattered light reliably from particle | grains, the light with a fixed light direction, a wavelength, and light intensity is preferable. Specifically, a laser, LED, etc. can be mentioned, for example. When using a laser, the type is not particularly limited, but an argon ion (Ar) laser, a helium-neon (He-Ne) laser, a die (dye) laser, a krypton (Cr) laser, a semiconductor laser, or a semiconductor laser and a wavelength. One or more solid lasers combined with conversion optical elements can be used in any combination.

  In the present technology, the light irradiation unit 2 may irradiate the microparticles flowing through the flow path with different axes. In the case of different axis irradiation, a plurality of spots are generated. However, if the flow rate is not stable, a time difference occurs between the spots, which adversely affects the measurement result. Therefore, by controlling the temperature of the sheath liquid to stabilize the flow velocity, this problem can be solved and the measurement accuracy can be improved.

(3) Detection unit 3
The detection unit 3 detects light from the fine particles.

  The detection unit 3 detects light components such as fluorescence, forward scattered light, side scattered light, and back scattered light generated from the fine particles in response to the light irradiation from the light irradiation unit 2 to the fine particles. These fluorescence and the necessary scattered light components are important light components for obtaining optical information (characteristics) of the microparticles.

  The type of the detection unit 3 is not particularly limited as long as it can detect light from fine particles, and a known photodetector can be freely selected and employed. For example, fluorescence measuring device, scattered light measuring device, transmitted light measuring device, reflected light measuring device, diffracted light measuring device, ultraviolet spectroscopic measuring device, infrared spectroscopic measuring device, Raman spectroscopic measuring device, FRET measuring device, FISH measuring device, In addition, various kinds of spectrum measuring devices, so-called multi-channel photodetectors in which a plurality of photodetectors are arranged in an array can be used alone or in combination of two or more.

  In the present technology, the detection unit 3 may include a light receiving element that receives light generated from the fine particles. Examples of the light receiving element include an area imaging element such as a CCD or a CMOS element, a PMT (Photomultiplier Tube), and a photodiode. In this case, the detection part 3 can also be comprised from the several light receiving element which has a different detection wavelength range. By configuring the detection unit 3 from a plurality of light receiving elements having different detection wavelength ranges, it is possible to measure the light intensity in a continuous wavelength range as a fluorescence spectrum. Specifically, for example, a PMT array or a photodiode array in which light receiving elements are arranged one-dimensionally, or a structure in which a plurality of independent detection channels such as a two-dimensional light receiving element such as a CCD or a CMOS are arranged.

(4) Sorting unit 4
The microparticle measurement apparatus 100 according to the present technology may further include a sorting unit 4 as necessary. The sorting unit 4 sorts out the target fine particles.

(4-1) Microchip M1
In the present technology, when the flow path is formed in the microchip M1, the sorting by the sorting unit 4 can be performed outside the microchip M1. Specifically, for example, as shown in FIG. 1 or 2, the sorting unit 4 includes a vibrating element 4a that generates a droplet, a deflecting plate 4b that changes a charged droplet in a desired direction, At least a collection container to collect. The charging unit 41 is separately defined in FIGS. 1 and 2, but is a part of the sorting unit 4, and performs charging based on a sorting control signal generated by the analyzing unit 5 described later.

  FIG. 5 is a schematic diagram illustrating an example of the configuration of the microchip M1 that can be used in the microparticle measurement apparatus 100 of FIG. 1 or 2, and FIG. 6 is applicable to the microparticle measurement apparatus 100 of FIG. It is a schematic diagram which shows an example of a structure of the orifice M11 of the microchip M1. 5A is a schematic top view, and FIG. 5B is a schematic cross-sectional view corresponding to the PP cross section in A. FIG. 6A is a top view, FIG. 6B is a cross-sectional view, and FIG. 6C is a front view. In addition, B of FIG. 6 respond | corresponds to the PP cross section in A of FIG.

  The microchip M1 is formed by bonding substrate layers M1a and M1b on which a sample channel M12 is formed. Formation of the sample flow path M12 on the substrate layers M1a and M1b can be performed by injection molding of a thermoplastic resin using a mold. As the thermoplastic resin, conventionally known plastics can be adopted as microchip materials such as polycarbonate, polymethyl methacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, and polydimethylsiloxane (PDMS).

  In the microchip M1, a sample introduction part M13 for introducing a sample containing microparticles, a sheath introduction part M14 for introducing a sheath liquid, and a sample flow path M12 for introducing a sample flow and joining with the sheath liquid are formed. After the sheath liquid introduced from the sheath introduction part M14 is divided and fed in two directions, the sample liquid is sandwiched from the two directions at the junction with the sample liquid introduced from the sample introduction part M13. Join the liquid. As a result, a three-dimensional laminar flow in which the sample liquid laminar flow is located at the center of the sheath liquid laminar flow is formed at the junction.

  M15 shown in FIG. 5A is for eliminating clogging and bubbles by applying a negative pressure in the sample channel M12 to temporarily reverse the flow when clogging or bubbles are generated in the sample channel M12. The suction flow path is shown. At one end of the suction flow path M15, a suction opening M151 connected to a negative pressure source such as a vacuum pump is formed. The other end of the suction channel M15 is connected to the sample channel M12 at the communication port M152.

  In the three-dimensional laminar flow, narrowing portions M161 (see FIG. 5) and M162 (A in FIG. 6) formed such that the area of the vertical cross section with respect to the liquid feeding direction gradually or gradually decreases from the upstream to the downstream of the liquid feeding direction. And B)), the laminar flow width is narrowed down. Thereafter, the three-dimensional laminar flow is discharged as a fluid stream from an orifice M11 provided at one end of the flow path.

  The fluid stream ejected from the orifice M11 is formed into droplets by the vibration element 4a applying vibration to the orifice M11. The orifice M11 opens in the direction of the end faces of the substrate layers M1a and M1b, and a notch M111 is provided between the opening position and the end face of the substrate layer. The notch M111 is formed by notching the substrate layers M1a and M1b between the opening position of the orifice M11 and the substrate end surface so that the diameter L1 of the notch M111 is larger than the opening diameter L2 of the orifice M11. (See C in FIG. 6).

  In the microchip M1, it is preferable that the diameter L1 of the notch M111 is formed to be twice or more larger than the opening diameter L2 of the orifice M11 so as not to hinder the movement of the droplet discharged from the orifice M11.

  In the microparticle measuring apparatus 100 of FIG. 1 or 2, the vibration element 4a generates a droplet by applying vibration to the orifice of the microchip M1. The charging unit 41 charges the liquid droplets discharged from the orifice of the microchip M1 positively or negatively based on the sorting control signal generated by the analyzing unit 5. Then, the charged droplets are sorted in a desired direction by a deflection plate (counter electrode) 4b to which a voltage is applied.

  In addition, the vibration element 4a is not specifically limited, A well-known thing can be selected freely and can be used, for example, a piezoelectric element etc. can be used. In addition, by adjusting the amount of liquid fed to the flow path, the diameter of the discharge port, the vibration frequency of the vibration element 4a, etc., the size of the droplet is adjusted to generate a droplet containing a certain amount of fine particles. Can do.

(4-2) Microchip M2
FIG. 7 is a schematic conceptual diagram showing a third embodiment of the microparticle measurement apparatus according to the present technology. In the present technology, when the flow path is formed in the microchip M2, the sorting by the sorting unit 4 may be performed in the microchip M2.

  The sample liquid containing fine particles is introduced from the sample liquid inlet M21 into the sample liquid flow path M22. Further, the sheath liquid is introduced from the sheath liquid inlet M23. The sheath liquid introduced from the sheath liquid inlet M23 is divided into two sheath liquid flow paths M24 and M24 and sent. The sample liquid flow path M22 and the sheath liquid flow paths M24 and M24 merge to form the main flow path M25. The sample liquid laminar flow sent through the sample liquid flow path M22 and the sheath liquid laminar flow sent through the sheath liquid flow paths M24 and M24 merge in the main flow path M25, and the sample liquid laminar flow becomes the sheath liquid. A sheath flow sandwiched between laminar flows is formed.

  8 indicates a detection region where excitation light is irradiated by the light irradiation unit 2 and fluorescence and scattered light are detected by the detection unit 3. The fine particles are sent to the detection region M25a in a state of being arranged in a line in the sheath flow formed in the main flow path M25, and are irradiated with excitation light from the light irradiation unit 2.

  The main flow path M25 is branched into three flow paths downstream of the detection region M25a. The main channel M25 communicates with three branch channels, a sorting channel M26 and waste channels M27 and M27, downstream of the detection region M25a. Among these, the sorting channel M26 is a channel into which fine particles determined to satisfy predetermined optical characteristics (hereinafter referred to as “target particles”) are taken. On the other hand, the fine particles determined not to satisfy the predetermined optical characteristics (hereinafter also referred to as “non-target particles”) are not taken into the sorting flow path M26, and any of the two waste flow paths M27 are used. Flows to either side.

  The target particles are taken into the sorting flow path M26 by generating a negative pressure in the sorting flow path M26 by the vibration element 4a such as a piezo element, and the sample liquid and the sheath containing the target particles using the negative pressure. This is performed by sucking the liquid into the sorting channel M26. The vibration element 4a is disposed in contact with the surface of the microchip M2, and is disposed at a position corresponding to the sorting flow path M26. More specifically, the vibration element 4a is disposed at a position corresponding to the pressure chamber M261 provided as a region where the inner space is expanded in the sorting flow path M26.

  The inner space of the pressure chamber M261 is expanded in the plane direction (width direction of the sorting flow path M26) as shown in FIG. 8, and at the same time in the cross-sectional direction (height of the sorting flow path M26 as shown in FIG. 10). (Direction) is also expanded. That is, the sorting flow path M26 is expanded in the width direction and the height direction in the pressure chamber M261. In other words, the sorting channel M26 is formed in the pressure chamber M261 so as to have a large vertical cross section with respect to the flow direction of the sample liquid and the sheath liquid.

  The vibration element 4a generates a stretching force with a change in applied voltage, and causes a pressure change in the sorting flow path M26 via the surface (contact surface) of the microchip M2. When a flow occurs in the sorting flow path M26 with a change in pressure in the sorting flow path M26, the volume in the sorting flow path M26 changes at the same time. The volume in the sorting flow path M26 changes until it reaches a volume defined by the amount of displacement of the vibration element 4a corresponding to the applied voltage. More specifically, the vibration element 4a presses the displacement plate 4a1 (see FIG. 10) constituting the pressure chamber M261 to keep the volume of the pressure chamber M261 small when the voltage element is applied and expanded. Yes. When the applied voltage decreases, the vibration element 4a generates a force in a contracting direction, and generates a negative pressure in the pressure chamber M261 by weakening the pressure on the displacement plate 4a1.

  In order to efficiently transmit the expansion / contraction force of the vibration element 4a into the pressure chamber M261, as shown in FIG. 10, the surface of the microchip M2 is recessed at a position corresponding to the pressure chamber M261, and the vibration element is inserted into the recess. It is preferable to arrange 4a. As a result, the displacement plate 4a1 serving as the contact surface of the vibration element 4a can be made thin, and the displacement plate 4a1 can be easily displaced by the change in the pressing force accompanying the expansion and contraction of the vibration element 4a, resulting in a volume change of the pressure chamber M261. it can.

  A symbol M256 in FIG. 10 indicates a communication port of the sorting channel M26 to the main channel M25. The target particles sent through the sheath flow formed in the main channel M25 are taken into the sorting channel M26 from the communication port M256.

  The microchip M2 is formed by laminating a substrate layer on which the main flow path M25 and the like are formed. Formation of the main channel M25 and the like on the substrate layer can be performed by injection molding of a thermoplastic resin using a mold. As the thermoplastic resin, a conventionally known plastic can be adopted as a microchip material such as polycarbonate, polymethyl methacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, and polydimethylsiloxane (PDMS).

  Note that the sorting unit 4 is not essential in the microparticle measurement device 100 according to the present technology, and the microparticle measurement device 100 according to the present technology does not perform sorting of the target microparticles, but only for analysis. It may be configured to stay.

(5) Analysis unit 5
The microparticle measurement apparatus 100 according to the present technology may further include an analysis unit 5 as necessary. The analysis unit 5 is connected to the detection unit 3 and analyzes the light detection value for the microparticles detected by the detection unit 3.

  In the analysis unit 5, for example, the detection value of the light received from the detection unit 3 is corrected, and the feature amount of each microparticle is calculated. Specifically, feature quantities indicating the size, form, internal structure, etc. of the microparticles are calculated from the detected values of the received fluorescence and scattered light. In addition, it is possible to make a sorting determination based on the calculated feature amount and a sorting condition received from the input unit in advance, and generate a sorting control signal.

  Note that the analysis unit 5 is not essential in the microparticle measurement device 100 according to the present technology, and based on the detection value of the light detected by the detection unit 3, the state of the microparticles using an external analysis device or the like Can also be analyzed. For example, the analysis unit 5 may be implemented by a personal computer or CPU, and is stored as a program in a hardware resource including a recording medium (for example, a nonvolatile memory (USB memory), HDD, CD, etc.), It is also possible to function by a personal computer or CPU. Moreover, the analysis part 5 may be connected to each part of the microparticle measuring apparatus 100 via a network.

(6) Storage unit 6
The microparticle measurement apparatus 100 according to the present technology may further include a storage unit 6 as necessary. The storage unit 6 stores all items related to the measurement such as the value detected by the detection unit 3, the feature amount calculated by the analysis unit 5, the sorting control signal, and the sorting condition input by the input unit. .

  Note that the storage unit 6 is not essential in the microparticle measurement device 100 according to the present technology, and an external storage device may be connected. As the storage unit 6, for example, a hard disk or the like can be used. Furthermore, the recording unit 6 may be connected to each unit of the fine particle measuring apparatus 100 via a network.

(7) Display unit 7
The microparticle measurement device 100 according to the present technology may further include a display unit 7 as necessary. The display unit 7 displays all items related to the measurement such as the value detected by the detection unit 3 and the feature amount calculated by the analysis unit 5. For example, the display unit 7 displays the feature amount for each microparticle calculated by the analysis unit 5 as a scattergram.

  Note that the display unit 7 is not essential in the microparticle measurement device 100 according to the present technology, and an external display device may be connected. For example, a display, a printer, or the like can be used as the display unit 7.

(8) Input unit 8
The microparticle measurement apparatus 100 according to the present technology may further include an input unit 8 as necessary. The input unit 8 is a part for a user such as an operator to operate. The user accesses each control unit through the input unit 8 and controls each unit of the microparticle measurement apparatus 100. For example, the input unit 8 sets a region of interest for the scattergram displayed on the display unit 7 and determines sorting conditions.

  Note that the input unit 8 is not essential in the microparticle measurement device 100 according to the present technology, and an external operation device may be connected. For example, a mouse, a keyboard, or the like can be used as the input unit 8.

(9) Control unit 9
The microparticle measurement apparatus 100 according to the present technology may further include a control unit 9 as necessary. The control unit 9 can control each unit of the fine particle measuring apparatus 100. Here, the control unit 9 is a concept different from the temperature control unit 11 described above.

  The control unit 9 may be arranged separately for each part of the microparticle measurement device 100 or may be provided outside the microparticle measurement device 100. For example, it may be implemented by a personal computer or CPU, and is stored as a program in a hardware resource including a recording medium (for example, a nonvolatile memory (USB memory), HDD, CD, etc.), and is executed by the personal computer or CPU. It is also possible to function. Moreover, the control part 9 may be connected with each part of the microparticle measuring apparatus 100 via a network.

(10) Insertion section 101
The microparticle measurement device 100 according to the present technology may further include an insertion unit 101 as necessary. When the flow path is formed on a substrate such as a microchip, the insertion unit 101 inserts the substrate into the microparticle measurement apparatus 100 and sets it.

(11) Sample feeding part 102
The microparticle measurement device 100 according to the present technology may further include a sample liquid feeding unit 102 as necessary. The sample liquid feeding unit 102 feeds the sample to the sample introduction unit via the tube. For example, the sample liquid feeding unit 102 sucks and feeds a sample from a test tube or well plate containing a sample via a nozzle, or applies pressure to a storage unit that can store a test tube containing a sample. To feed the sample.

(12) Drainage unit 103
The microparticle measurement device 100 according to the present technology may further include a drainage unit 103 as necessary. The drainage part 103 is a part where the drainage is sent through a tube. The drainage unit 103 includes, for example, a drainage tank.

2. Microparticle measurement method The microparticle measurement method according to the present technology includes at least a sheath liquid introduction step, a light irradiation step, and a detection step. Further, other steps may be performed as necessary. Hereinafter, each step will be described in detail.

(1) Sheath liquid introduction process In the sheath liquid introduction process, the sheath liquid is introduced from the sheath liquid reservoir into the flow path where the sample liquid containing microparticles and the sheath liquid merge. In this step, the temperature of the sheath liquid is controlled. The specific method performed in this step is the same as the method performed in the sheath liquid introduction unit 1 of the microparticle measurement device 100 described above, and therefore the description thereof is omitted here.

(2) Light irradiation process In a light irradiation process, light is irradiated to the microparticle which flows through the said flow path. The specific method performed in this step is the same as the method performed in the light irradiation unit 2 of the fine particle measuring apparatus 100 described above, and thus the description thereof is omitted here.

(3) Detection step In the detection step, light from the fine particles is detected. The specific method performed in this step is the same as the method performed in the detection unit 3 of the microparticle measurement apparatus 100 described above, and thus description thereof is omitted here.

Note that the present technology may have the following configurations.
(1)
A sheath liquid introduction section for introducing the sheath liquid from the sheath liquid storage section to the flow path where the sample liquid containing the microparticles and the sheath liquid merge;
A light irradiation unit for irradiating light to the microparticles flowing through the flow path;
A detection unit for detecting light from the microparticles;
Comprising at least
The sheath liquid introduction unit includes a temperature control unit that controls the temperature of the sheath liquid.
(2)
The sheath liquid introduction part connects the sheath liquid storage part and the flow path via a tube,
The microparticle measurement apparatus according to (1), wherein the tube includes at least one temperature sensor.
(3)
The fine particle measuring apparatus according to (2), wherein the temperature sensor is provided so as to be in contact with the surface of the tube.
(4)
The temperature sensor is the microparticle measurement device according to (2) or (3), which is provided in the vicinity of the flow path.
(5)
The fine particle measuring device according to any one of (2) to (4), wherein the temperature sensor is connected to the temperature control unit through a circuit.
(6)
The said sheath liquid introducing | transducing part is a microparticle measuring apparatus in any one of (2) to (5) further equipped with the temperature control unit which adjusts the temperature of the said sheath liquid.
(7)
The said temperature control unit is a microparticle measuring apparatus as described in (6) provided with a temperature control element, a metal member, and the temperature test | inspection part which test | inspects the temperature of the said metal member.
(8)
The fine particle measuring apparatus according to (7), wherein the metal member is provided so as to sandwich the tube.
(9)
The fine particle measuring apparatus according to (7) or (8), wherein the temperature control element and the temperature inspection unit are connected to the temperature control unit through a circuit.
(10)
The microparticle measurement apparatus according to (9), wherein the temperature control unit controls the temperature control element by PI control.
(11)
The said temperature sensor is a microparticle measuring device in any one of (6) to (10) provided between the said sheath liquid storage part and the said temperature control unit.
(12)
The said light irradiation part is a microparticle measuring apparatus in any one of (1) to (11) which irradiates light to the microparticle which flows through the said flow path from a different axis.
(13)
The microparticle measurement apparatus according to any one of (1) to (12), further including a sorting unit that sorts target microparticles.
(14)
The flow path is formed in a microchip,
The fine particle measuring apparatus according to (13), wherein the sorting by the sorting unit is performed outside the microchip.
(15)
The flow path is formed in a microchip,
The microparticle measurement apparatus according to (13), wherein the sorting by the sorting unit is performed in the microchip.
(16)
A sheath liquid introducing step of introducing the sheath liquid from the sheath liquid reservoir to the flow path where the sample liquid containing the microparticles and the sheath liquid merge;
A light irradiation step of irradiating light to the microparticles flowing through the flow path;
A detection step of detecting light from the microparticles;
At least
A method for measuring microparticles, wherein the temperature of the sheath liquid is controlled in the sheath liquid introduction step.

DESCRIPTION OF SYMBOLS 100: Fine particle measuring device 1: Sheath liquid introducing | transducing part 11: Temperature control part 12: Temperature control unit 121: Temperature control element 122: Metal member 123: Temperature test | inspection part 124: Heat sink 125: Fan 111a: Provided near the said flow path Temperature sensor 111b: temperature sensor 110 provided between the sheath liquid storage unit 110 and the temperature control unit 12: sheath liquid storage unit 2: light irradiation unit 3: detection unit 4: sorting unit 5: analysis unit 6 : Storage unit 7: Display unit 8: Input unit 9: Control unit 101: Insertion unit 102: Sample liquid feeding unit 103: Drainage unit M1, M2: Microchip

Claims (16)

A sheath liquid introduction section for introducing the sheath liquid from the sheath liquid storage section to the flow path where the sample liquid containing the microparticles and the sheath liquid merge;
A light irradiation unit for irradiating light to the microparticles flowing through the flow path;
A detection unit for detecting light from the microparticles;
Comprising at least
The sheath liquid introduction unit includes a temperature control unit that controls the temperature of the sheath liquid. The sheath liquid introduction part connects the sheath liquid storage part and the flow path via a tube,
The microparticle measurement apparatus according to claim 1, wherein the tube includes at least one temperature sensor.   The microparticle measurement apparatus according to claim 2, wherein the temperature sensor is provided so as to be in contact with a surface of the tube.   The microparticle measurement apparatus according to claim 2, wherein the temperature sensor is provided in the vicinity of the flow path.   The microparticle measurement apparatus according to claim 2, wherein the temperature sensor is connected to the temperature control unit through a circuit.   The microparticle measurement apparatus according to claim 2, wherein the sheath liquid introduction unit further includes a temperature adjustment unit that adjusts a temperature of the sheath liquid.   The fine particle measuring apparatus according to claim 6, wherein the temperature control unit includes a temperature control element, a metal member, and a temperature inspection unit that inspects the temperature of the metal member.   The microparticle measurement apparatus according to claim 7, wherein the metal member is provided so as to sandwich the tube.   The microparticle measurement apparatus according to claim 7, wherein the temperature control element and the temperature inspection unit are connected to the temperature control unit through a circuit.   The microparticle measuring apparatus according to claim 9, wherein the temperature control unit controls the temperature control element by PI control.   The microparticle measurement apparatus according to claim 6, wherein the temperature sensor is provided between the sheath liquid storage unit and the temperature control unit.   The microparticle measuring apparatus according to claim 1, wherein the light irradiation unit irradiates light on the microparticles flowing through the flow path from different axes.   The microparticle measurement apparatus according to claim 1, further comprising a sorting unit that sorts target microparticles. The flow path is formed in a microchip,
The fine particle measuring device according to claim 13, wherein the sorting by the sorting unit is performed outside the microchip. The flow path is formed in a microchip,
The fine particle measuring apparatus according to claim 13, wherein the sorting by the sorting unit is performed in the microchip. A sheath liquid introducing step of introducing the sheath liquid from the sheath liquid reservoir to the flow path where the sample liquid containing the microparticles and the sheath liquid merge;
A light irradiation step of irradiating light to the microparticles flowing through the flow path;
A detection step of detecting light from the microparticles;
At least
A method for measuring microparticles, wherein the temperature of the sheath liquid is controlled in the sheath liquid introduction step.