JP4976209B2 - Specimen identification and dispensing apparatus and specimen identification and dispensing method - Google Patents

Description

  The present invention relates to a specimen identification and dispensing apparatus and a specimen identification and dispensing method. In particular, after the specimen is identified based on the optical information of the separated specimen, the specimen is dispensed to a predetermined specimen dispensing position without causing contamination (contamination) and affecting the specimen. The present invention relates to a specimen identification and dispensing apparatus and a specimen identification and dispensing method that can shorten the processing time of a dispensing operation.

A liquid in which a specimen such as a cell (microscopic object) such as a cell is dispersed in the liquid flows in the capillary tube, and light from the light source is irradiated onto this liquid stream, so that optical information on the specimen in the liquid stream ( It has been proposed to identify specimens by measuring fluorescence information. After the specimen is identified, the specimen is subjected to ultrasonic vibration in the dispensing unit to form a droplet, and is charged with, for example, several hundred volts. Then, by applying a voltage of several thousand volts from the deflecting plate, the drop position of each droplet is divided into a plus pole side and a minus pole side, and dispensed into an arbitrary container (well) of the dispensing unit.
Tatsuro Yamashita, Shinichiro Niwa, Cell Engineering Vol. 16, no. 10 p1532-1541, 1997

  However, when a specimen such as a cell is dispensed in this way, high-frequency vibration or a high voltage of several thousand volts is applied to a small droplet containing the specimen during dispensing. For example, when the specimen is a living cell, The mortality rate of the specimen after injection is high, and even if the specimen is alive, there is no guarantee that the specimen is in a normal state, and there is a problem that it will adversely affect stem cell culture and differentiation. there were. In addition, since the dispensing of specimens requires the formation of small droplets, it touches a lot of air in addition to ultrasonic waves and electric charges, causing contamination of the droplets containing the specimen and damage to the specimen. There is concern.

  In addition, the dispensing operation of a plurality of samples in the dispensing unit includes a mechanical movement operation (mechanical movement operation) when selecting a well to be dispensed from among the wells, and the dispensing operation requires time. There was also a problem that it took. The speed in the dispensing operation is generally about 1 sort / sec. Therefore, for example, in the case of a target cell dispensing operation in which the total number of cells is 100,000 and the presence rate is 0.01%, the processing time required for the dispensing operation of all the cells is as long as 100,000 seconds (28 h). I was sorry. Here, the presence rate is a ratio of the number of target cells to be sorted among all cells.

  Therefore, the present invention has been made to solve the above-described problems, and does not cause contamination after sample identification and does not affect the sample. An object of the present invention is to provide a specimen identification and dispensing apparatus and a specimen identification and dispensing method capable of dispensing at a specimen dispensing position and shortening the processing time of the dispensing operation.

  The following invention is provided to solve the above-mentioned conventional problems.

The sample identification and dispensing apparatus according to the first aspect of the present invention is a sample identification and separation unit for collecting a target sample to be sampled from a sample to be measured dispersed in a sample liquid flowing in a flow path. An injection device that measures optical information of the specimen by irradiating the specimen with excitation light, and identifies the specimen based on the measured optical information of the specimen, and the identification A dispensing part for dispensing a preparative solution in which one or a plurality of the specimens identified by the part are dispersed into a dispensing target site via a nozzle; and the specimen in the sample liquid in which the specimen is dispersed The number of target and non-target samples to be sorted in the sorting solution can be adjusted to the desired number based on the concentration of the sample solution that is the number concentration of the sample and the volume of the sorting solution. includes a concentration adjusting unit such, the flow path of the nozzle, the Characterized in that widened as compared with the flow path in another portion.

The sample identification and dispensing apparatus according to the second aspect of the present invention is the sample identification and dispensing apparatus according to the first aspect of the present invention, wherein the amount of the preparative solution is the flow rate of the sample liquid and the nozzle. characterized in that it is adjusted on the basis of the injection time of the dispensing the dispensing unit and the operating time of the nozzle for dispensing into the target site, and preparative said fraction to the dispensing target site solution via .

  The sample identification / dispensing device according to the third aspect of the present invention is the sample identification / dispensing device according to the first or second aspect of the present invention, wherein the dispensing unit presets the fractionation solution. Dispensing to the same part to be dispensed the same number of times.

The sample identification and dispensing apparatus according to the fourth aspect of the present invention is the sample identification and dispensing apparatus according to any one of the first to third aspects of the present invention, wherein the identification unit is based on a plurality of identification setting conditions. Te, identifying the specimen, the dispensing unit includes wherein based on the plurality of identification setting conditions for identifying the specimen in the identification part is dispensed into a plurality of the dispensing target site To do.

  The sample identification and dispensing apparatus according to a fifth aspect of the present invention is the sample identification and dispensing apparatus according to any one of the first to fourth aspects of the present invention, wherein the dispensing unit It is possible to move three-dimensionally.

The sample identification / dispensing device according to the sixth aspect of the present invention is the sample identification / dispensing device according to any one of the first to fifth aspects of the present invention. a formed well, said in the well are accommodated accommodating liquid for containing the specimen, the solution preparative the fraction containing the analyte to be discharged from the tip opening of the nozzle, droplet from the nozzle The liquid is dispensed in contact with the stored liquid in the well without forming a liquid crystal.

  The sample identification / dispensing device according to the seventh aspect of the present invention is the sample identification / dispensing device according to the sixth aspect of the present invention, wherein the sorting solution containing the sample is formed in a hemispherical shape at the tip of the nozzle. The specimen is a cell, and the accommodation liquid in the well is a culture solution.

  The specimen identifying and dispensing apparatus according to the eighth aspect of the present invention is the specimen identifying and dispensing apparatus according to the sixth or seventh aspect of the present invention, wherein the portion of the nozzle forming the tip opening is the The outer diameter is tapered toward the opening of the tip.

A sample identification and dispensing apparatus according to a ninth aspect of the present invention is the sample identification and dispensing apparatus according to any one of the first to eighth aspects of the present invention, wherein the sample is separated and separated from the identification unit. A supply unit configured to distribute and supply the sample to the sample solution, and the sample flow path formed by the identification unit and the nozzle distributes the sample to the portion to be dispensed Until now, it is characterized by being formed in a straight line.

A sample identification and dispensing apparatus according to a tenth aspect of the present invention is the sample identification and dispensing apparatus according to any one of the first to ninth aspects of the present invention, wherein the sample identification and dispensing apparatus includes the target sample including the target sample. A resupply unit that supplies the liquid as the sample liquid to the supply unit, and adjusting the abundance ratio that is a ratio of the total number of the target samples to the total number of the samples in the sample liquid in the supply unit Features.

The sample identification and dispensing apparatus according to an eleventh aspect of the present invention is the sample identification and dispensing apparatus according to any one of the first to tenth aspects of the present invention, wherein the concentration of the sample liquid containing the target sample is a predetermined value. When within the concentration region, the concentration adjusting unit adjusts the number of the specimens in the sorting solution to one, and the dispensing unit has one of the above-described dispensing target sites. The preparative solution in which the target specimen is dispersed is dispensed.

The specimen identification and dispensing method according to the first aspect of the present invention is a specimen identification and separation method for fractionating a target specimen to be sorted out of specimens to be measured dispersed in a sample liquid flowing in a flow path. (A) a supplying step in which the specimen is separated and supplied to the sample liquid, and (b) optical information of the specimen is measured by irradiating the specimen with excitation light. And an identification step for identifying the sample based on the measured optical information of the sample, and (c) one or more of the samples in the dispersed solution identified in the identification step (b). A concentration adjusting step for adjusting the number; and (d) the preparative solution identified by the identifying step (b) and the number adjusted by the concentration adjusting step (c ) in the flow path in the identifying step (b). min through a nozzle having a flow passage widened compared A concentration step (c), wherein the concentration adjusting step (c) includes a sample solution concentration that is the number concentration of the analyte in the sample solution in which the analyte is dispersed, and the dispensing based on the liquid amount of the solution, and adjusting the number of the number and the non-target specimen of the target analyte of preparative interest in the solution preparative said fraction to a desired number.

The specimen identification and dispensing method according to the second aspect of the present invention is the specimen identification and dispensing method according to the first aspect of the present invention, wherein the amount of the preparative solution is the flow rate of the sample liquid and the nozzle. based on the dispensing mechanical operation time of the dispensing process for dispensing to the target site (c), and injection time of the preparative solution of the preparative solution into the dispensation target site via It is characterized by being adjusted.

  The specimen identification and dispensing method according to the third aspect of the present invention is the specimen identification and dispensing method according to the first or second aspect of the present invention, wherein the dispensing step (d) Dispensing to the same portion to be dispensed a set number of times.

The specimen identification and dispensing method according to the fourth aspect of the present invention is the specimen identification and dispensing method according to any one of the first to third aspects of the present invention, wherein the identification step (b) includes a plurality of identification settings. based on the conditions, and identifying the specimen, the dispensing step (d), based on the plurality of identification setting conditions for identifying the specimen in the identification step (b), a plurality of the dispensing target Dispensing into parts.

  The sample identification and dispensing method according to the fifth aspect of the present invention is the sample identification and dispensing method according to any one of the first to fourth aspects of the present invention, wherein (e) the dispensing target containing the target sample is provided. A ratio of the total number of the target specimens to the total number of the specimens in the sample liquid of the supply step (a), comprising a resupply step of supplying the liquid in the site as the sample liquid to the supply step (a) It is characterized by adjusting the abundance ratio.

  The sample identification and dispensing method according to the sixth aspect of the present invention is the sample identification and dispensing method according to any one of the first to fifth aspects of the present invention, wherein the concentration of the sample liquid containing the target sample is a predetermined value. When within the concentration range, the concentration adjusting step (c) adjusts the number of the specimens in the sorting solution to one, and the dispensing step (d) includes one portion to be dispensed. And the dispensing solution in which one target sample is dispersed is dispensed.

  According to the present invention, it is possible to dispense a specimen at a dispensing position without forming a droplet from the nozzle after identifying the specimen contained in the liquid bleeding from the tip of the dispensing nozzle. Not only is it possible to improve the viability of living cells without damaging sensitive living cells such as, but it also has an extremely positive effect on stem cell culture and differentiation, and is extremely important for the practical application of stem cell regenerative medicine. Play a role. Further, it is possible to quickly dispense the sample to a predetermined sample dispensing position without causing contamination and without affecting the sample.

  Concentrate the sample solution step by step, that is, increase the presence rate of the target sample, adjust the concentration of the sample in the appropriate sample solution (sample solution concentration), and then target samples one by one By dispensing, it is possible to shorten the time required for the mechanical moving operation which takes time in the dispensing operation. Therefore, the dispensing work time can be shortened.

  An embodiment of the present invention will be described with reference to the drawings. In addition, the embodiment described below is for explanation, and does not limit the scope of the present invention. Accordingly, those skilled in the art can employ embodiments in which each or all of these elements are replaced by equivalents thereof, and these embodiments are also included in the scope of the present invention.

  FIG. 1 is a perspective view showing a preferred embodiment of the specimen identifying and dispensing apparatus of the present invention. FIG. 2 shows the sample identification and dispensing apparatus of FIG. 1 in more detail.

  In FIG. 1, a sample identification and dispensing apparatus 10 includes a sample supply unit 11, an optical measurement device 12 as a sample identification unit, a sample dispensing unit 13, and a control unit including a sample concentration adjustment unit 400. 100 and a specimen re-supply unit (not shown). The specimen is also called a test minute object or sample, and a plurality of specimens are dispersed in the liquid. The specimen identifying and dispensing apparatus 10 is also referred to as a flow cytometer.

  The specimen supply unit 11 shown in FIG. 1 separates the specimens S and SR and supplies them together with the liquid to the optical measurement device 12 side via the tube 14. This liquid forms a sample stream for sending the specimens S and SR. The optical measuring device 12 receives the fluorescence information of the specimens S and SR by irradiating the specimens S and SR passing through the capillary as the flow path along the Z1 direction with, for example, excitation light from a laser light source. The specimens S and SR are identified.

  The dispensing unit 13 illustrated in FIG. 1 injects a plurality of identified samples S into a well (an example of a container) W at an arbitrary dispensing target position. Here, the sample S is a target sample to be dispensed into the well W, and the sample SR is a non-target sample to be discarded. The control unit 100 in FIG. 1 adjusts the concentration of the specimen, analyzes the fluorescence information measured by the optical measurement device 12, and controls the dispensing unit 13.

  The specimen supply unit 11 illustrated in FIG. 1 supplies the specimens S and SR together with the sample liquid 20 to the optical measurement device 12 side through the tube 14 as illustrated in FIG.

  In the optical measuring device 12, a sample flow including the specimens S and SR and a sheath flow that wraps the sample flow flow in the capillary 21, and the excitation light L from the laser light source passes through the specimens S and SR. For example, the specimens S and SR generate fluorescence information. The fluorescence information is received by the light receiving unit 42, and the fluorescence information generated by the specimens S and SR is analyzed by the control unit 100 in FIG. The specimens S and SR that have been analyzed by the control unit do not form droplets through the nozzle 30 and are dispensed into an arbitrary well W or discarded into the waste liquid tank 300 by the dispensing unit 13 in FIG. It has come to be.

  The concentration adjusting unit 400 of the control unit 100 shown in FIG. 1 is configured to supply the sample solution concentration that is the number concentration of the specimens S and SR in the sample solution 20 supplied from the supply unit 11 to the optical measurement device 12 and the nozzle 30. By adjusting the amount of the preparative solution dispensed to any well W of the dispensing unit 13, an arbitrary number of specimens S and SR are dispensed to the well W. Here, the preparative solution dispensed into the well W includes at least one specimen S based on the result of analyzing the fluorescence information measured by the optical measuring device 12.

  The amount of the sample solution is determined based on the flow rate of the sample solution 20 supplied by the supply unit 11, the operation time of the dispensing unit 13, the operation time of the nozzle 30, and the injection time of the solution to the well W. Adjusted based on. Here, in FIG. 1, the dispensing operation is performed by three-dimensionally moving the dispensing unit 13 with respect to the nozzle 30, and thus the operation time of the dispensing unit 13 is listed as an adjustment factor. When 30 moves, the moving time of the nozzle 30 becomes an adjustment factor instead of the operation time of the dispensing unit 13. Moreover, when the nozzle 30 and the dispensing part 13 move together, it becomes an adjustment element of the moving time of the nozzle 30 and the dispensing part 13.

  Further, the control unit 100 shown in FIG. 1 controls the dispensing unit 13 to dispense the specimens S and SR into an arbitrary well W through the nozzle 30 in a dispensing operation, or a waste liquid tank. It can be disposed of in 300. At this time, the same well W can be dispensed a predetermined number of times. Further, the samples S and SR can be identified based on a plurality of identification setting conditions, and can be dispensed into a plurality of wells W based on the identification setting conditions. For example, when the setting of the excitation light L from the laser light source is a voltage of 1 to 2 V and the setting is 2 when the voltage of 3 to 5 V is a setting 2, the sample S measured when the setting is set to the well W1 Alternatively, the sample S measured when setting 2 can be dispensed into the well W2.

  The resupply unit supplies the samples S and SR in the well W of the dispensing unit 13 in FIG. 1 to the supply unit 11 in FIG. 1 using a nozzle or the like.

  FIG. 3 shows a shape example of the capillary 21 and the nozzle 30. The connecting portion 32 of the tube 14 shown in FIG. 3 is connected to one end portion 33 of the capillary 21, and the other end portion 34 of the capillary 21 is connected to one end portion 35 of the nozzle 30. The capillary 21 and the nozzle 30 form a linear flow path for flowing the specimen S.

  As shown by a range R shown in FIG. 3, the capillary 21 is a hollow member whose inner diameter is constant along the axial direction CL, and is made of linear transparent glass or plastic. The cross-sectional shape of the capillary 21 is, for example, a rectangle.

  An optical fiber 40 is disposed corresponding to the capillary 21. The excitation light L generated by the laser light source 41 is irradiated to the specimen S passing through the capillary 21 by the optical fiber 40.

  Next, an example of the shape of the nozzle 30 shown in FIG. 3 will be described.

  The nozzle 30 is a substantially cylindrical member and has one end portion 35, the other end portion 50, and an intermediate portion 51. The nozzle 30 has a nozzle passage portion 52 along the axial direction CL, and the nozzle passage portion 52 includes an inlet portion 53, an intermediate passage 54, and a tip opening portion 55. The inner diameter D1 of the inlet portion 53 is set smaller than the inner diameter D2 of the intermediate passage 54 and the inner diameter D2 of the tip opening 55. As a result, a trumpet-shaped portion 56 that extends along the Z1 direction is formed between the inlet portion 53 and the intermediate passage 54, and the intermediate passage 54 and the tip opening 55 become a passage portion having a constant inner diameter D2. Yes.

  By adopting such a shape of the nozzle 30, the flow velocity of the liquid containing the sample S passing from the inlet portion 53 toward the intermediate passage 54 is decreased because the inner diameter gradually increases in the trumpet-shaped portion 56. be able to. For this reason, even if the specimen S such as a living cell flows through the nozzle 30, it is possible to prevent the specimen S from being damaged by the pressure generated by the flow velocity.

  In the range R of the capillary 21 in FIG. 3, for example, one side of the cross section is 20 mm, and the length F of the nozzle 30 is, for example, 70 mm to 80 mm. The inner diameter D2 of the nozzle 30 is, for example, 400 μm, and the inner dimensions of the capillary 21 are, for example, 150 μm in the vertical direction and 300 μm in the horizontal direction.

  As shown in FIGS. 3 and 4, the tapered portion 60 of the other end portion 50 of the nozzle 30 is formed to be tapered toward the outlet portion 55. In addition, as shown in FIG. 4, the tip opening 55 of the tapered portion 60 of the nozzle 30 causes the hemispherical liquid 150 containing the identified specimen S to come into contact with the surface 71 of the culture solution 70. The specimen S is dispensed into the culture solution 70 in the well W such that the tip opening 55 of the 30 tapered portion 60 does not directly contact the culture solution 70 in the well W. Thereby, when the taper part 60 of the nozzle 30 enters the well W of the plate 200 of the dispensing part 13, the size of the part where the taper part 60 enters the well W is tapered. Compared to the case of no nozzle, it can be reduced, and the occurrence of contamination (contamination) with respect to the specimen and the culture solution can be prevented. Further, the liquid 150 can be prevented from dropping as a droplet (for example, the droplet 1002 in FIG. 17).

  Further, as shown in FIGS. 1 and 2, in the capillary 21 and the nozzle 30 of the optical measurement device 12, the liquid containing the sample S flows straight along the Z1 direction in a straight line, so the liquid containing the sample S There is little change in the flow rate, and the flow rate can be stabilized.

  Next, the dispensing unit 13 will be described with reference to FIG.

  The dispensing unit 13 in FIG. 1 includes a culture plate (hereinafter referred to as a plate) 200, a movement operation unit 250, and a waste liquid tank 300.

  The plate 200 is disposed in an XY plane formed by the X-axis direction and the Y-axis direction. The plate 200 has a plurality of wells W, and the plurality of wells W are arranged in a matrix at a predetermined pitch along the X-axis direction and the Y-axis direction. As shown in FIG. 4, a culture solution 70 that is an example of a storage liquid is stored in each well W.

  The waste liquid tank 300 shown in FIG. 1 is disposed on the upper side of the plate 200 so as to be parallel to the plate 200 along the Y-axis direction.

  An example of the structure of the waste liquid tank 300 is shown in FIG. 5, and the waste liquid tank 300 is a bowl-shaped member having a U-shaped cross section. One end portion 301 of the waste liquid tank 300 is fixed to a holder 302, and the holder 302 is movable along the X-axis direction along the guide bar 303. The guide bar 303 is fixed to the plate 200 along the X-axis direction at the side of the plate 200.

  As a device for moving and positioning the waste liquid tank 300 in the X-axis direction, for example, a mechanism using a motor and a feed screw rotated by the motor can be employed. The operation of the motor M in this case is controlled by a command from the control unit 100. The feed screw is arranged in parallel to the guide bar 303, and the waste screw tank 300 can be moved and positioned in the X-axis direction by rotating the feed screw by the operation of the motor M.

  As shown in FIG. 5, the waste liquid tank 300 is used for discarding the liquid 150 containing the specimen discharged from the nozzle 30 into the waste liquid 160 as necessary. At this time, when the liquid 150 discharged from the tip opening 55 of the nozzle 30 includes an unnecessary specimen SR, the liquid 150 bleeding from the nozzle tip is in contact with the surface of the waste liquid 160. To be discarded.

  As shown in FIG. 5, the waste liquid 160 is gradually discharged from the waste liquid tank 300 by the operation of the tubing pump 310, but the waste liquid tank 300 is normally filled with the waste liquid 160. The waste liquid is reliably sucked by the tubing pump 310 so as not to overflow from the waste liquid tank 300. Thereby, the waste liquid does not leak from the waste liquid tank 300.

  In contrast, if the nozzle 30 is inserted into the waste liquid 160 as in the comparative example shown in FIG. 6, the nozzle 30 directly contacts the waste liquid 160. For this reason, if an attempt is made to dispense the sample S into the well W using the nozzle 30 thereafter, the culture solution in the well W may cause contamination (contamination) through the nozzle 300. However, in the embodiment of the present invention, since the tip outlet portion 55 of the nozzle 30 does not directly contact the waste liquid 160 of the waste liquid tank 300, contamination to the culture solution in the well W can be prevented. .

  1 can move and position the plate 200 along the X-axis direction, the Y-axis direction, and the Z-axis direction. The X-axis direction (first direction), the Y-axis direction (second direction), and the Z-axis direction (third direction) are orthogonal to each other, and the Z-axis direction is the vertical direction in FIG. As the movement operation unit 250, for example, a three-axis movement table such as a commonly used XYZ table can be used.

  Next, an operation example of the dispensing unit 13 will be described with reference to FIG. 1 and FIGS.

  FIG. 1 shows a standby state of the sample identification / separation apparatus 10, and the nozzle 30 is provided in the Z1 direction on the lower side of the optical measurement apparatus 12, and the nozzle 30 is a waste liquid tank 300 of the dispensing unit 13. It is in the position corresponding to. The waste liquid tank 300 is located at the standby position P1 at the center of the plate 200 in the X-axis direction.

  FIG. 7 shows a state in which the waste liquid tank 300 descends from the standby position P1 of FIG. 1 and is retracted, and the plate 200 and the waste liquid tank 300 are integrated as a result of the movement operation unit 250 operating according to a command from the control unit 100. Thus, it is lowered in the Z1 direction and positioned at the retracted position P2. For this reason, the waste liquid tank 300 is in a lower position away from the nozzle 30.

  FIG. 8 shows a state where the waste liquid tank 300 is further retracted in the X1 direction from the retreat position P2 of FIG. In contrast, it moves in the X1 direction (left direction in FIG. 8) and is positioned at the retracted position P3. As a result, the waste liquid tank 300 is located away from the nozzle 30.

  Next, FIG. 9 shows a state in which the plate 200 and the waste liquid tank 300 are raised in the Z2 direction, and the plate 200 and the waste liquid tank 300 are moved in the Z2 direction when the movement operation unit 250 is operated according to a command from the control unit 100. And is positioned at the dispensing position P4. As a result, as shown in FIG. 4, the liquid 150 exuding from the tip opening 55 of the nozzle 30 comes into contact with the culture solution 70 of the selected well W, so that the liquid 150 containing the specimen S is in the well W. Will be dispensed. At this time, the other end portion (lower end portion) 50 of the nozzle 30 is tapered, and the other end portion 50 is not in direct contact with the culture solution 70, so that the culture solution 70 and the sample S in the well W are not contacted. In contrast, it is possible to reliably prevent contamination from entering from the nozzle 30 side.

  FIG. 10 shows a state where the plate 200 and the waste liquid tank 300 are lowered in the Z1 direction again, and the well W is moved down by the command of the control unit 100, so that the well W moves away from the nozzle 30. Is positioned.

  FIG. 11 shows a state in which the waste liquid tank 300 is again positioned at the standby position P <b> 6 below the nozzle 30. When the moving operation unit 250 is operated according to a command from the control unit 100, the waste liquid tank 300 moves in the X2 direction and moves toward the standby position P6. At the same time, the plate 200 moves in the X1 direction opposite to the X2 direction. Is moved along the arrangement pitch of the wells. As a result, the nozzle 30 is relatively positioned above the arbitrary well W1 at the next position. Since the waste liquid tank 300 is moved in the X2 direction and the plate 200 is moved in the X1 direction by the well arrangement pitch, the time required for relatively positioning the nozzle 30 above the next candidate well W1 is increased. It can be shortened.

  FIG. 12 shows a standby state in which the plate 200 and the waste liquid tank 300 are lifted in the Z2 direction, and the plate 200 and the waste liquid tank 300 are lifted in the Z2 direction when the moving operation unit 250 is operated according to a command from the control unit 100. Then, it is positioned at the standby position P7.

  By performing such a series of operations, the nozzle 30 can dispense the liquid 150 to wells at arbitrary positions on the plate 200. In FIG. 11, in order to dispense the liquid 150 to the well W2 instead of the well W1, the moving operation unit 250 is operated according to a command from the control unit 100, so that the plate 200 and the waste liquid tank 300 are aligned in the Y1 direction. It moves by the arrangement pitch and is positioned. The waste liquid tank 300 moves together with the plate 200 only by the movement in the Z-axis direction. However, when the waste liquid tank 300 moves in the X-axis direction, it can be moved and positioned separately from the plate 200. Instead of the nozzle 30 moving, the position of the nozzle 30 is fixed. Instead, the plate 200 of the dispensing unit 13 and the unit of the waste liquid tank 300 are moved in the X-axis direction, the Y-axis direction, and the Z-axis of the moving operation unit 250. It moves along the axial direction.

  FIG. 17 shows a structure of a dispensing unit 1000 used conventionally as a comparative example.

  After the specimen 1002 is identified, the specimen 1002 applies ultrasonic vibrations in the dispensing unit 1000 to form droplets, and gives an electric charge at several hundred volts, for example. Then, by applying a voltage of several thousand volts from the deflecting plate, the drop position of each droplet is divided into the positive electrode side and the negative electrode side and dispensed into the well 1003 of the dispensing unit. Since a high voltage such as high-frequency vibration or several thousand volts is applied to the specimen 1002 during this dispensing, for example, when the specimen 1002 is a living cell, the mortality rate of the specimen after dispensing is high and the specimen 1002 is alive. Even if it does, it cannot be guaranteed that the specimen 1002 is in a normal state.

  On the other hand, by using the specimen identification and dispensing apparatus 10 according to the embodiment of the present invention, such a charge is not applied or a voltage is not applied, and the plate of the dispensing unit 13 is applied to the nozzle 30. The 200 side can move in the X-axis direction, the Y-axis direction, and the Z-axis direction, and the sample can be quickly dispensed to a predetermined sample dispensing position without adversely affecting the sample. In addition, since the position of the nozzle 30 is fixed and the plate 200 side moves, there is no inconvenience that the specimen leaks from the nozzle 30 compared to the case where the nozzle moves.

  Also, as shown in FIG. 2, the flow path of the sample formed by the optical measuring device 12 that is the identification unit and the nozzle 30 is formed in a straight line, so that the sample as in the comparative example shown in FIG. Since the flow rate of the specimen S can be stabilized as compared with the case where the S optical measurement portion 1500 is configured by the curved tube 1530, the accuracy during the optical measurement of the specimen can be increased.

  In the sample identification and dispensing apparatus 10 according to the embodiment of the present invention, the sample S that is the measurement target dispersed in the liquid flowing in the capillary 21 serving as the flow path is irradiated with the excitation light L, whereby the sample S An optical measurement device 12 that is an identification unit for measuring and identifying optical information, and a dispensing unit 13 for dispensing the identified specimen S to the well W that is a dispensing target site via the nozzle 30 ,have. The dispensing unit 13 can move three-dimensionally with respect to the identification unit 12 and the nozzle 30. Thus, after the sample S is identified, the sample 30 can be quickly dispensed to a predetermined sample dispensing position without moving the nozzle 30 side and without adversely affecting the sample S.

  In the specimen identification and dispensing apparatus 10 according to the embodiment of the present invention, a plurality of dispensing target sites are wells W formed on the plate 200, and culture as an accommodation liquid for containing the specimen S in the wells W. The liquid 70 is contained, and the liquid containing the sample S discharged from the tip opening 55 of the nozzle 30 is dispensed in a state in contact with the culture solution 70 in the well W. Thereby, the nozzle 30 can dispense the liquid containing the sample S without directly contacting the culture solution 70, and does not cause contamination to the sample and the culture solution after the sample is identified. The sample can be quickly dispensed to a predetermined sample dispensing position without affecting the sample.

  In the specimen identifying and dispensing apparatus 10 according to the embodiment of the present invention, the liquid containing the specimen is hemispherical, the specimen is a cell, and the accommodation liquid in the well is a culture solution. As a result, the cell does not cause contamination after the sample is identified with respect to the culture solution 70, and quickly dispenses the cell to a predetermined sample dispensing position without affecting the cell. Can do.

  In the sample identification and dispensing apparatus 10 according to the embodiment of the present invention, the part forming the tip opening of the nozzle is tapered toward the tip opening. As a result, it is possible to reduce the contact of the nozzle 30 with the liquid contained in the well W as much as possible, so that no contamination occurs after the specimen is identified and the specimen is not affected. A sample can be quickly dispensed to a predetermined sample dispensing position.

  In the sample identification and dispensing device 10 of the embodiment of the present invention, the flow path of the nozzle 30 is wider than the flow path in the optical measurement device 12. Thereby, when the liquid containing the sample S flows from the flow path in the optical measuring device 12 into the nozzle, the flow rate of the liquid containing the sample S can be slowed down, so that the flow rate can be stabilized. The optical information of S can be obtained accurately.

  The sample identification and dispensing apparatus 10 according to the embodiment of the present invention includes a supply unit 11 for separating the sample S and supplying the sample S to the optical measurement device 12, and is formed by the optical measurement device 12 and the nozzle 30. The flow path of the specimen S to be formed is formed in a straight line. Thereby, the flow rate of the liquid containing the specimen S can be stabilized, and the optical information of the specimen S can be obtained accurately.

  FIG. 13 shows another operation example in which the waste liquid tank 300 returns to the standby position after being retracted.

  In FIG. 13A, the tip of the nozzle 30 is inserted into the waste liquid in the waste liquid tank 300. In FIG. After the front end is retracted from the waste liquid tank 300, it is retracted upward and rearward. Accordingly, the waste liquid tank 300 can be retracted backward without hitting the nozzle 30.

  In FIG. 13C, the sample S can be dispensed by the plate 200 rising in the Z2 direction and the tip of the nozzle 30 being inserted into the well W. In FIG. 13D, the plate 200 is lowered in the Z1 direction, and the tip end of the slur 30 is separated from the inside of the well W. In FIG. 13E, the waste liquid tank 300 is moved in the G direction and is placed in the waste liquid tank 300. When the tip of the nozzle 30 is inserted again, the waste liquid tank 300 returns to the standby position. At the same time, the plate 200 is moved so that the specimen can be dispensed to the next well.

  Next, a dispensing process for dispensing a target sample will be described with reference to FIGS.

  FIG. 14 is a diagram for explaining a change in the presence rate of the target specimen due to the dispensing process. As shown in FIG. 14, in the dispensing process, the presence rate M0% of the target sample supplied from the supply unit 11 in FIG. 1 is calculated in the well W of the dispensing unit 13 in FIG. The presence rate of the sample S to be dispensed is increased to M1%.

  By repeating this dispensing operation a plurality of times through the resupply unit, the presence rate of the target sample to be dispensed in the well W of the dispensing unit 13 in FIG. Purpose of dispensing into the well W of the dispensing unit 13 (when the result of the n-time dispensing operation: the concentration of the sample solution supplied from the supply unit 11 in FIG. 1 is within a predetermined concentration range) The target abundance Mn% is set to 100%, and one target sample is dispensed into the well W one by one.

  That is, after the second time, a plurality of samples including at least one target sample dispensed in the well W of the dispensing unit 13 in FIG. 1 are supplied to the supply unit 11 in FIG. 1 in the next dispensing operation. Then, the sequential dispensing operation is executed m times.

  Thereby, in the specimen identification and dispensing apparatus 10 of the embodiment of the present invention, the sample liquid is concentrated stepwise, that is, the target specimen is increased in the presence rate, and the specimen concentration in the appropriate sample liquid is increased. (Sample solution concentration) and then dispensing target samples one by one, thereby reducing the number of operation operations of the dispensing unit 13 of FIG. Thus, the dispensing process time can be shortened.

  Next, the results of an example of the dispensing process by the sample identification and dispensing apparatus 10 according to the embodiment of the present invention will be described with reference to FIGS. 15 and 16.

  In this example, the dispensing operation was performed twice, and a dispensing process for dispensing 10 target cells out of a total of 100,000 cells was performed. FIG. 15 is a diagram for explaining the embodiment. FIG. 16 shows the result of the dispensing process of the example.

  As shown in FIG. 15 and FIG. 16, first, the concentration at which a total of 100,000 cells containing 10 target cells are dispersed is 1000 cells / μl from the supply unit 11 of FIG. The amount of the sample solution was 100 μl, and 75 cells each containing at least one target cell were dispensed into 10 wells W.

  Accordingly, in the supply unit 11 of FIG. 1, the target cell abundance rate of 0.01% is reduced to 1.33%. became. The time required for the first dispensing operation was 11.1 min. At this time, the liquid contained in the well W was 50 μl in advance, and the liquid volume in the well W was 80 μl.

  Next, in the second dispensing operation, a total of 750 cells containing 10 target cells dispensed in the first time (the cell volume was dispersed in 80 μl with a sample solution concentration of 9.38 cells / μl). 1) was supplied from the supply unit 11 in FIG. 1, and one target cell was dispensed into each of the ten wells W. Thereby, in the supply part 11 of FIG. 1, the presence rate of the target cell is 1.33%, and the presence rate of the target cell dispensed in the well W of the dispensing part 13 in FIG. 1 is 100%. . The time required for the second dispensing operation was 8.9 min.

  As a result, it was found that in the dispensing process for a total of 100,000 cells containing 10 target cells, the processing time that conventionally took 28 hours was 20 min, and the processing time was significantly reduced.

  By the way, this invention is not limited to the said embodiment, A various modified example is employable.

  For example, the light receiving unit 42 shown in FIG. 1 is arranged at a position opposite to the optical fiber 40 with the capillary 21 in between, but is not limited to this, and is located on the side of the capillary 21 (in the direction perpendicular to the plane of FIG. 1). (Position along).

  The capillary 21 shown in FIGS. 1 and 2 is a hollow member having a rectangular cross section, for example, but may have other cross sectional shapes.

  The dispensing unit 13 is a plate, but is not limited to a plate, and may be a tube, a dish, or the like.

  Further, the re-supply unit does not need to be provided in the sample identification and dispensing device as a mechanism, and it is sufficient that the dispensed sample can be supplied again to the optical measurement device 12 via the supply unit 13.

  In addition, signals such as scattered light, transmitted light, and fluorescence information obtained from, for example, cells that are the specimen S can be acquired using the light receiving unit 42.

  The transparent member is not limited to a glass plate, but may be another material such as a plastic plate as long as it is transparent.

  The nozzle 30 may be inclined with respect to the waste liquid tank 300 instead of being perpendicular to the illustrated example.

  In the present invention, the excitation light can also be referred to as measurement light or irradiation light.

  The optical measuring device of the present invention is used in fields requiring examination, analysis and analysis of biopolymers of genes, immune systems, proteins, amino acids, and sugars, such as engineering, food, agriculture, fishery processing, etc. It can be applied to all fields such as fields, hygiene, health, immunity, plague, genetic fields such as heredity, and science fields such as chemistry or biology.

It is a perspective view which shows preferable embodiment of the sample identification dispensing apparatus of this invention. It is a figure which shows a supply part, an identification part, and a dispensing part. It is sectional drawing which shows an identification part and a nozzle. It is sectional drawing which shows the front-end | tip part and well of a nozzle. It is a figure which shows the state which brought the nozzle close to the waste liquid tank of a dispensing part. It is a figure which shows the state which inserted the nozzle in the waste liquid tank of a dispensing part. It is a figure which shows the state which the waste liquid tank retracted from the standby position P1 of FIG. FIG. 8 is a view showing a state where the waste liquid tank is further retracted in the X1 direction from the retreat position P2 of FIG. It is a figure which shows the state which the plate rose to Z2 direction. It is a figure which shows the state which the plate fell in the Z1 direction again. It is a figure which shows the state by which the waste liquid tank was positioned in the standby position P6 of the lower part of a nozzle again. It is a figure which shows the standby state which the plate and the waste liquid tank rose to Z2 direction. It is a figure which shows another example of an operation | movement which returns to a standby position, after a waste liquid tank evacuates. It is a figure for demonstrating the change of the presence rate of the target sample by a dispensing process. It is a figure for demonstrating an Example. It is a result of the dispensing process of an Example. It is a figure which shows the structure of the dispensing part 1000 used conventionally as a comparative example. It is a figure which shows the comparative example comprised by the tube 1530 in which the optical measurement part of the test substance was curved.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Sample identification dispensing apparatus 11 Supply part 12 Optical measuring device (identification part)
13 Dispensing part 21 Capillary (an example of a flow path)
30 nozzles 41 laser light source (an example of a light source)
55 Tip opening 60 Tapered portion of nozzle 100 Control unit 150 Liquid 160 Waste liquid 200 Plate (culture plate)
250 Moving operation unit 300 Waste liquid tank 400 Concentration adjusting unit S Sample (sample)
W Well

Claims (17)

A sample identification and dispensing device for dispensing a target sample to be sampled from a sample to be measured dispersed in a sample liquid flowing in a flow path,
An identification unit for measuring the optical information of the specimen by irradiating the specimen with excitation light, and identifying the specimen based on the measured optical information of the specimen;
A dispensing unit for dispensing a preparative solution in which one or a plurality of the specimens identified by the identification unit are dispersed to a dispensing target site via a nozzle;
The number of target specimens to be sorted in the preparative solution based on the sample liquid concentration, which is the number concentration of the specimen in the sample liquid in which the specimen is dispersed, and the amount of the preparative solution And a concentration adjusting unit capable of adjusting the number of non-target specimens to a desired number,
Equipped with a,
The specimen identification and dispensing apparatus , wherein a flow path of the nozzle is wider than a flow path in the identification section . Amount of liquid in the preparative solution, of the sample liquid flow, the dispensing unit and the operating time of the nozzle for dispensing into the dispensation target site through the nozzle, and to the dispensing target site The specimen identifying and dispensing apparatus according to claim 1, wherein the specimen identifying and dispensing apparatus is adjusted based on an injection time of the sorting solution.   The sample identification and dispensing apparatus according to claim 1 or 2, wherein the dispensing unit dispenses the sorting solution into the same portion to be dispensed a predetermined number of times. The identification unit identifies the sample based on a plurality of identification setting conditions,
The dispensing portion, based on the plurality of identification setting conditions for identifying the specimen in the identification part, any of claims 1 to 3, characterized in that dispensing a plurality of the dispensing target site The specimen identification and dispensing apparatus according to claim 1.   5. The specimen identifying and dispensing apparatus according to claim 1, wherein the dispensing unit is three-dimensionally movable with respect to the nozzle. The portion to be dispensed is a plurality of wells formed on the plate, the containing liquid for containing the sample is stored in the well, and the sample discharged from the tip opening of the nozzle is stored in the well. 6. The dispensing solution according to claim 1, wherein the sorting solution is dispensed in a state of being in contact with the stored liquid in the well without forming a droplet from the nozzle. Sample identification and dispensing device.   The preparative solution containing the specimen is formed in a hemispherical shape at the tip of the nozzle, the specimen is a cell, and the accommodation liquid in the well is a culture solution. Sample identification and dispensing device.   8. The specimen identification and dispensing apparatus according to claim 6, wherein a portion of the nozzle forming the tip opening is formed so that an outer diameter thereof is tapered toward the tip opening. . A supply unit for separating and supplying the sample to the sample liquid by separating the sample and supplying the sample liquid;
The flow path of the sample formed by the identification unit and the nozzle is formed in a straight line until the sample is dispensed to the dispensing target site. 9. The specimen identification and dispensing device according to any one of items 8 to 9. A resupply part for supplying the liquid in the dispensing target site containing the target specimen to the supply part as the sample liquid;
10. The sample identification dispensing according to claim 1 , wherein a presence rate that is a ratio of a total number of the target samples to a total number of the samples in the sample solution in the supply unit is adjusted. apparatus. When the concentration of the sample solution containing the target specimen is within a predetermined concentration range,
The concentration adjusting unit adjusts the number of the specimens in the preparative solution to one;
11. The dispenser according to claim 1, wherein the dispensing unit dispenses the sorting solution in which one target sample is dispersed in one dispensing target site. 11. Sample identification and dispensing device. A sample identification and dispensing method for dispensing a target sample to be sampled from a sample to be measured dispersed in a sample liquid flowing in a flow path,
(A) a supply step of separating and supplying the sample to be dispersed in the sample liquid;
(B) an identification step of measuring the optical information of the specimen by irradiating the specimen with excitation light, and identifying the specimen based on the measured optical information of the specimen;
(C) a concentration adjusting step of adjusting the number of the specimens in one or a plurality of dispersed solutions identified in the identifying step (b);
(D) A flow path that is broadened in comparison with the flow path in the identification step (b) is used for the preparative solution identified in the identification step (b) and whose number is adjusted in the concentration adjustment step (c). A dispensing process for dispensing to a dispensing target site via a nozzle having
With
In the concentration adjusting step (c), the concentration in the preparative solution is determined based on the sample liquid concentration, which is the number concentration of the sample in the sample liquid in which the sample is dispersed, and the amount of the preparative solution. A sample identification and dispensing method, wherein the number of target samples to be taken and the number of non-target samples are adjusted to desired numbers . The amount of the sample solution is the flow rate of the sample solution, the mechanical operation time of the dispensing step (c) for dispensing to the dispensing target site via the nozzle, and the dispensing target. The specimen identification and dispensing method according to claim 12, wherein the specimen identification and dispensing method is adjusted based on an injection time of the fractionation solution into the site . The sample identification and dispensing method according to claim 12 or 13, wherein the dispensing step (d) dispenses the fractionated solution to the same portion to be dispensed a predetermined number of times. . The identification step (b) identifies the specimen based on a plurality of identification setting conditions,
The dispensing step (d) is characterized in that the dispensing step (d) dispenses the plurality of portions to be dispensed based on the plurality of identification setting conditions for identifying the specimen in the identification step (b). Item 15. The specimen identification and dispensing method according to any one of Items 12 to 14 . (E) a resupply step of supplying the liquid in the dispensing target site containing the target specimen as the sample liquid to the supply step (a),
The specimen according to any one of claims 12 to 15, wherein a presence rate that is a ratio of a total number of the target specimens to a total number of the specimens in the sample solution in the supply step (a) is adjusted. Identification dispensing method. When the concentration of the sample solution containing the target specimen is within a predetermined concentration range,
The concentration adjustment step (c) adjusts the number of the specimens in the preparative solution to one,
The dispensing step (d), one of the dispensing target site, one any one of the said preparative solution of the desired analyte dispersed claim 12, wherein the dispensing 16 The specimen identification and dispensing method described in 1.

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