JP2012247224A - Specimen dilution apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a diluting apparatus that can handle samples (analytes) in various forms other than liquid, has a simple structure, and is inexpensive.
A diluting solution supply unit for diluting a sample, a diluting unit for loading the sample and diluting the sample with the diluting solution, a diluting solution feeding and a diluting sample diluted by the diluting unit A liquid feeding part capable of sucking and discharging, the liquid feeding part having a suction port and a discharge port, a liquid feeding means capable of feeding the diluted liquid from the suction port to the discharge port, and a liquid The above-described problem is solved by a diluting device that includes a suspendable diaphragm and a suction / discharge port, and one or more suction / discharge means capable of sucking and discharging the liquid in the diaphragm.
[Selection] Figure 3

Description

  The present invention relates to a sample (specimen) dilution apparatus. In particular, the present invention relates to a sample (specimen) dilution apparatus for preparing a sample (diluted specimen) to be introduced into an analyzer such as a liquid chromatograph apparatus.

  When separating and analyzing components contained in a biological sample such as blood and urine, a liquid chromatograph (FIG. 1) is often used. However, while these biological samples contain a wide variety of components, the amount of components (target components) to be separated and analyzed is often very small. For this reason, when a biological sample is directly introduced into an analysis column provided in a liquid chromatograph apparatus, the analysis column is often damaged or the separation and analysis of a target component is disturbed. Therefore, when performing separation analysis of a target component in a biological sample using a liquid chromatograph apparatus, the biological sample is usually diluted before being introduced into the liquid chromatograph apparatus. In addition, when the biological sample is blood and the target component in the blood cell is separated and analyzed, it is necessary to hemolyze with a hemolyzing agent or hemolyzed before being introduced into the liquid chromatograph apparatus.

  Many methods for diluting a biological sample or adding a hemolytic agent to a blood sample have been commercialized or disclosed so far. As an example, using a dispensing device equipped with a syringe pump and a needle, a small amount of air, a predetermined amount of diluent, a small amount of air, a predetermined amount of sample liquid are sucked into the needle in this order, and then dispensed into the dilution container. Then, there is a method of preparing a diluted sample by stirring by suction / discharge with a needle in the dilution container (Patent Document 1). As another example, the diluted solution is stored in advance in a dilution port capable of holding a certain amount of liquid, and the diluted sample is prepared by discharging and stirring the liquid sample (specimen) to the port by a dispensing means. There are methods (Patent Documents 2 and 3). Note that the sample diluted by the above-described method is introduced into the analysis column using a dispensing means such as a syringe. As another method, a method is also disclosed in which a liquid sample (specimen) and a diluent are mixed in a pipe and dilution is performed online by natural diffusion (Patent Document 4).

JP 2011-013045 A JP 2002-031626 A JP-A-9-178719 JP 2001-343371 A

  As described above, many methods for diluting a biological sample (specimen) and adding a hemolytic agent to a blood sample have been commercialized or disclosed. However, in any of the methods described above, the biological sample (specimen) is a liquid sample accommodated in a blood collection tube, a sample cup, or the like, and the specimen in another mode (for example, a mode in which the biological sample is absorbed by a filter paper piece) is used. It is difficult to handle. Further, since the means (unit) used in the above-described method is constituted by an expensive syringe unit or the like, the operation is complicated and the price tends to be expensive.

  Accordingly, an object of the present invention is to provide a diluting apparatus that can handle various types of samples (specimens) other than liquid, has a simple structure, and is inexpensive.

  This invention made | formed in view of the said subject includes the following aspects.

The first aspect is
A diluent supply section for diluting the specimen;
A dilution section for loading a specimen and diluting the specimen with the diluent;
A liquid feeding section capable of aspirating and discharging the diluted specimen and the diluted specimen diluted by the dilution section;
A diluting device comprising:
A liquid feeding means having a suction port and a discharge port, and capable of feeding the diluent from the suction port to the discharge port;
One or more suction / discharge means having a diaphragm capable of holding liquid and a suction / discharge port and capable of sucking / discharging the liquid in the diaphragm;
It is the said dilution apparatus provided with.

The second aspect is
The liquid feeding part is
A liquid feeding means having a suction port and a discharge port, and capable of feeding the diluent from the suction port to the discharge port;
A first and second suction / discharge means having a diaphragm capable of holding liquid and two suction / discharge ports, and capable of sucking and discharging the liquid in the diaphragm;
With
The discharge port of the liquid feeding means communicates with one of the suction discharge ports of the first suction / discharge means,
The other of the suction and discharge ports of the first suction and discharge means communicates with one of the suction and discharge ports of the second suction and discharge means,
The other of the suction and discharge ports of the second suction and discharge means and the diluting portion can communicate with each other;
It is a dilution apparatus as described in said 1st aspect.

The third aspect is
The dilution section is
A dilution port that holds the diluted solution or diluted sample and can load the sample,
A drain port capable of discharging a diluted solution or a diluted specimen;
The dilution device according to the first or second aspect, wherein the drain port is provided on an outer peripheral portion of the dilution port.

In the fourth aspect,
The dilution section is
A dilution port that holds the diluted solution or diluted sample and can load the sample,
A drain port provided on an outer peripheral portion of the dilution port and capable of discharging a diluted solution or a diluted specimen;
A first flow path for communicating the bottom of the dilution port with one of the suction and discharge ports of the suction and discharge means;
A second flow path for communicating the side surface portion of the dilution port with the first flow path;
The dilution apparatus according to the first or second aspect, wherein a check valve is provided in each of the first flow path and the second flow path.

  A fifth aspect is the dilution apparatus according to any one of the first to fourth aspects, wherein the liquid feeding means is a diaphragm pump.

  Hereinafter, the present invention will be described in detail.

A schematic view of an example of the diluting apparatus of the present invention is shown in FIG. 2, and a detailed view is shown in FIG. The dilution apparatus of the present invention is
A supply part of a diluent (12) for diluting the specimen;
A dilution section (14) for loading the specimen and diluting the specimen with the diluent (12);
A liquid feeding section (15) capable of aspirating and discharging a diluted specimen diluted with the diluent (12) and diluted by the dilution section (14);
It has. In the present invention, the diluted solution refers to a liquid added to the specimen in order to reduce the concentration of the target component and other components contained in the biological sample (specimen). In addition, when the biological sample (specimen) is blood, a liquid containing a hemolytic agent that is added to the blood specimen in order to separate and analyze the target component in the blood cells also corresponds to the diluent of the present invention.

First, in the diluting device of the present invention, the liquid feeding part will be described in detail. The liquid feeding section (15) provided in the dilution apparatus shown in FIG.
A liquid feeding means (17) having a suction port and a discharge port and capable of feeding a diluent from the suction port to the discharge port;
Two suction / discharge means (18a, 18b) having a diaphragm (41) capable of holding liquid and two suction / discharge ports, and capable of sucking and discharging the liquid in the diaphragm (41);
With
The discharge port of the liquid feeding means (17) communicates with one of the suction discharge ports of the suction discharge means (18a),
The other of the suction and discharge ports of the suction and discharge means (18a) communicates with one of the suction and discharge ports of the suction and discharge means (18b),
The other of the suction / discharge ports of the suction / discharge means (18b) can communicate with the dilution section (14).

An example of the liquid feeding means (17) in FIG. 3 is a simple pump capable of feeding the diluent in one direction (from the suction port to the discharge port) by the check valves (21a, 21b). A typical example of a simple pump is an electromagnetic diaphragm pump that has been commercialized in recent years. The electromagnetic diaphragm pump
A diaphragm (41) made of a flexible material such as rubber, which can hold liquid;
A piston (42) joined to a diaphragm (41);
An electromagnetic coil (45) for driving the piston (42) in one direction;
A spring (43) for driving the piston (42) in the opposite direction;
An adjustment screw (44) for determining the travel distance of the piston (42);
When the power to the electromagnetic coil (45) is turned on / off, the diaphragm (41) is deformed and the diluting liquid can be sent in one direction (from the suction port to the discharge port) ( FIG. 4a). The liquid feeding means (17) is not limited to the electromagnetic diaphragm pump described above, but may be a pump capable of feeding the diluting liquid in one direction (from the suction port to the discharge port) by a check valve (21a, 21b) or the like. That's fine. As an example, a diaphragm pump (FIG. 8b) that drives a piston (diaphragm) by a motor or a piezoelectric element, a tube pump represented by a peristaltic pump (FIG. 8c), and the like can be given.

As an example of the suction discharge means (18a, 18b) in FIG.
A diaphragm (41) made of a flexible material such as rubber, which can hold liquid;
A piston (42) joined to the diaphragm (41);
An electromagnetic coil (45) for driving the piston (42) in one direction;
A spring (43) for driving the piston (42) in the opposite direction;
An adjustment screw (44) for determining the drive distance of the piston (42);
The diaphragm (41) is deformed by turning on / off the power supply to the electromagnetic coil (45), and the liquid in the diaphragm (41) can be moved (FIG. 4b). The suction / discharge means shown in FIG. 4b is preferable because it has a structure and appearance very similar to those of the electromagnetic diaphragm pump (FIG. 4a) described above and is inexpensive in price. The capacity of the diaphragm (41) included in the suction / discharge means (18a) and the capacity of the diaphragm (41) included in the suction / discharge means (18b) may be the same capacity or different capacity. .

  The discharge port of the liquid feeding means (17) communicates with one of the suction / discharge ports of the suction / discharge means (18a), and the other of the suction / discharge ports of the suction / discharge means (18a) and the suction / discharge means (18b) The liquid in the liquid feeding part (that is, the liquid feeding part in which the liquid feeding means (17), the suction / discharge means (18a) and the suction / discharge means (18b) are connected in series) communicated with one of the suction and discharge ports. The flow will be described in detail with reference to FIGS.

(A) Cleaning step (FIGS. 5 (a-1) and (a-2))
When the diluting liquid is fed by the liquid feeding means (17), it is necessary to pass through the two suction / discharge means (18a, 18b) provided downstream. When the suction / discharge means (18a, 18b) is in the form shown in FIG. 4b, the power to the electromagnetic coil (45) is turned on, the piston (42) is moved upward, and the diaphragm (41) is inflated. The suction / discharge means (18a, 18b) can be in a penetrating state. In this state, the liquid supply means (17) is driven (specifically, the power supply is periodically turned ON / OFF), so that the diluted liquid is supplied to the liquid supply means (17) and the suction / discharge means (18a). Then, the liquid is fed in the order of the suction / discharge means (18b) and the diluting part (14), and the diluting liquid overflows from the diluting part (14). Replacement with liquid is performed.

(B) Discharging process (FIG. 5 (b))
In a state where the liquid feeding means (17) is stopped (that is, a state where the diaphragm is deflated), the power to the electromagnetic coil provided in the suction / discharge means (18a, 18b) is turned off to move the piston downward, so that the diaphragm (41) The dilute solution for the diaphragm (41) volume is discharged by making it deflated. Since the liquid feeding means (17) side is closed by the check valve (21a) on the discharge port side of the liquid feeding means (17), the diluted liquid is discharged only to the dilution section (14) side.

(C) Sample loading step (FIG. 6 (c))
After the specimen is loaded in the diluting section (14), the liquid feeding means (17) is stopped and the power to the electromagnetic coil provided in the suction / discharge means (18a, 18b) is turned on to move the piston upward. By making the diaphragm (41) swelled, a diluted solution (diluted sample) containing the sample is aspirated in the diaphragm (41). Since the liquid feed means (17) side is closed by the check valve (21a) on the discharge port side of the liquid feed means (17), the diaphragm (41) included in the suction discharge means (18a, 18b) is closed. Only diluted specimens are aspirated.

(D) Dilution step (FIGS. 6 (d-1) and (d-2))
By periodically turning on / off the power supply to the electromagnetic coil provided in the suction / discharge means (18b), the sample diluent for the diaphragm (41) volume provided in the suction / discharge means (18b) is sucked and discharged. The sample is diluted by reciprocating the liquid by the volume of the diaphragm.

  (D) After the diluting step, the diluted specimen in the diluting section is once held, and then an automatic dispensing means (Fig. 2a) using a microsyringe or the like from above the dilution port or an automatic dispensing means (Fig. 2) By extracting according to 2b), it can be introduced into a subsequent analysis means (for example, an analytical column). It is also possible to connect the injection valve to the flow path connecting the dilution section and the liquid feeding section and introduce the diluted specimen into the analysis means online (not shown).

  In FIGS. 5 and 6, the liquid feeding means (17) and the suction / discharge means (18a, 18b) have a structure in which the two suction / discharge ports are opened with the power turned on (generally normally closed). The operation in the case of the means (refer to FIG. 4a and FIG. 4b) has been described, but the structure in which the two suction outlets are opened with the power off (generally normally open) The liquid supply means (17) and the suction / discharge means (18a, 18b) may be provided. In this case, the power ON / OFF operation is reversed.

  When sample dilution is performed using the diluting apparatus shown in FIG. 3, the suction / discharge states of the liquid feeding means (17) and the suction / discharge means (18a, 18b), and the dilution port (31) provided in the dilution section to be described later A diagram summarizing the liquid level position is shown in FIG. In FIG. 7, symbols assigned to the respective steps correspond to the symbols in FIGS.

The liquid feeding means (17) and one or more suction / discharge means (18) included in the diluting device of the present invention include:
(A) Arrangement as individual means, each connected by external piping (FIG. 8),
May be,
(B) A mode (FIGS. 9 and 10) aggregated in the form of one manifold (61),
However, it is preferable to employ the aspect (B) in that the dilution apparatus can be saved in space. In the dilution apparatus shown in FIGS. 9 and 10, the liquid feeding means (17) is composed of a block including the suction / discharge means (18c) and a block including the check valve (21).

An example of the diluting unit provided in the diluting device of the present invention is shown in FIG. 11a. The dilution part shown in FIG.
A dilution port (31) that holds a fixed amount of the diluted solution or diluted sample and is capable of loading the sample,
A drain receiver (32) provided on the outer periphery of the dilution port (31) for receiving the diluted solution or the diluted specimen overflowing from the dilution port;
A drain port (33) provided on the outer periphery of the dilution port (31) and capable of discharging the diluted solution or the diluted specimen accumulated in the drain receiver (32) out of the system;
A flow path provided at the bottom of the dilution port (31) and a flow path (34) through which the diluted solution or diluted sample enters and exits;
It has. The outer shape of the dilution port (31) may be any shape as long as the overflowing diluted solution flows smoothly in the direction of the drain groove, and may be a simple cone or truncated cone shape shown in FIG. It may be a shape.

  FIG. 12 shows the flow of liquid in the dilution section shown in FIG. 11a during the dilution process and the cleaning process. When the amount of the diluent sucked and discharged from the liquid feeding part is less than or equal to the capacity of the dilution port (31), when the diluent is discharged from the liquid feeding part, the diluent is discharged from the bottom of the dilution port (31). When the dilution liquid is accumulated in the dilution port (31) (FIG. 12a) and the dilution liquid is sucked from the liquid feeding section, the dilution liquid is sucked from the bottom of the dilution port (31) to the liquid feeding section (FIG. 12b). Therefore, when the suction / discharge operation is repeated, the diluent reciprocates around the dilution port (31). Therefore, the specimen (dilution specimen preparation) can be diluted by loading the specimen that needs to be diluted to the dilution port (31) and repeating the aspiration and discharge operation. On the other hand, when the amount of the diluted liquid discharged from the liquid feeding part is equal to or larger than the capacity of the dilution port (31), the diluted liquid flowing in from the bottom part (34) of the dilution port (31) is accumulated in the dilution port (31). The diluted liquid that has exceeded the capacity and has overflowed is discharged from the drain port (33) through the drain receiver (32) (FIG. 12c). For example, when washing the dilution port (31), the dilution liquid is continuously discharged from the liquid feeding unit, and the dilution port (31) is overflowed.

  FIG. 13 shows the progress of specimen dilution (preparation of diluted specimen) by the dilution section shown in FIG. 11a. When a liquid sample is loaded on the dilution port (31) (FIG. 13a), the natural diffusion of the sample proceeds near the surface in the dilution port (31), while the concentration near the bottom remains low (FIG. 13b). Therefore, the sample dilution is gradually advanced from the bottom of the dilution port (31) by repeating the suction and discharge operation by the liquid feeding unit (FIGS. 13c to 13e).

FIG. 11b shows another example of the dilution section provided in the dilution apparatus of the present invention. The dilution part shown in FIG.
A dilution port (31) that holds a fixed amount of the diluted solution or diluted sample and is capable of loading the sample,
A drain receiver (32) provided on the outer periphery of the dilution port (31) for receiving the diluted solution or the diluted specimen overflowing from the dilution port;
A drain port (33) provided on the outer periphery of the dilution port (31) and capable of discharging the diluted solution or the diluted specimen accumulated in the drain receiver (32) out of the system;
A first flow path (34a) provided at the bottom of the dilution port (31) for entering and exiting the liquid feeding portion and the diluted solution or diluted specimen;
A second channel (34b) for communicating the side surface of the dilution port (31) with the first channel (34a);
The first flow path (34a) and the second flow path (34b) have check valves (21a, 21b), respectively. Of the first flow path (34a), the flow path from the branch to the second flow path (34b) to the dilution port (31) is connected from the dilution port (31) to the liquid feeding section by the check valve (21b). The second flow path (34b) can only send liquid from the liquid feeding part to the dilution port (31) by the check valve (21a). The outer shape of the dilution port (31) may be any shape as long as the overflowing diluted solution flows smoothly in the direction of the drain groove as in the dilution portion shown in FIG. 11a. The simple cone or truncated cone shape shown in FIG. It may be any other shape.

  FIG. 14 shows the flow of the liquid when the diluted liquid is sucked / discharged from the liquid feeding section to the dilution section shown in FIG. 11b. The dilution liquid discharged from the liquid feeding section is discharged from the side surface of the dilution port (31) through the second flow path (34b) branched from the first flow path (34a) (FIG. 14a). On the other hand, when the diluent is sucked from the liquid feeding section, the diluent is sucked from the bottom of the dilution port (31) through the first channel (34a) to the liquid feeding section (FIG. 14b). When the amount of diluent sucked and discharged from the liquid feeding part is less than or equal to the capacity of the dilution port (31), when the dilution liquid is discharged from the liquid feeding part, the dilution liquid is discharged from the side part of the dilution port (31). When the liquid is collected in the dilution port (31) and sucked from the liquid feeding part, the liquid is sucked from the bottom of the dilution port (31). Therefore, when the operation of sucking and discharging is repeated, the diluent is circulated around the dilution port (31), the first channel (34a), and the second channel (34b). Therefore, the specimen (dilution specimen preparation) can be diluted by loading the specimen that needs to be diluted to the dilution port (31) and repeating the aspiration and discharge operation. On the other hand, when the amount of the diluted liquid discharged from the liquid feeding part is equal to or larger than the capacity of the dilution port (31), the diluted liquid discharged from the side surface of the dilution port (31) is accumulated in the dilution port (31), Overflowing, the diluted liquid is discharged out of the system from the drain port (33) via the drain receiver (32) (FIG. 14c). For example, when the dilution port (31) is washed, the dilution liquid is continuously discharged from the liquid feeding section, and the dilution port (31) is overflowed.

  FIG. 15 shows the progress of specimen dilution (preparation of diluted specimen) by the dilution section shown in FIG. 11b. When a liquid sample is loaded on the dilution port (31) (FIG. 15a), the natural diffusion of the sample proceeds near the surface in the dilution port (31), while the concentration near the bottom remains low (FIG. 15b). In the dilution section shown in FIG. 15b, the liquid in the dilution port (31) is sucked from the bottom of the dilution port (31). Therefore, the diluent containing the low-concentration specimen flows through the first flow path (34a) via the check valve (21b) and is sucked into the liquid feeding section (FIG. 15c). On the other hand, when the specimen diluent is discharged, the diluent containing the low-concentration specimen described above flows through the second flow path (34b) via the check valve (21a) and enters the dilution port (31). It discharges from a side part (FIG. 15d). By the suction and discharge operation, the bottom of the dilution port (31) becomes a diluted solution containing a relatively high concentration sample, and the surface portion becomes a diluted solution containing a relatively low concentration sample (FIG. 15e). By repeating this operation, the diffusion zone expands while the interface between the suction and descending liquid and the side discharge liquid formed one after another circulates through the second flow path and the first flow path. The sample dilution can be efficiently advanced.

In the diluting apparatus of the present invention, a sample that requires dilution is loaded on the dilution port (31), but the mode of the loaded sample is not particularly limited. As an example,
(A) A method of directly loading a liquid specimen (7) using a dispenser (16a) such as a syringe or pipette (FIG. 16a),
(B) A method of aspirating the liquid specimen (7) using the capillary (16b) and loading the specimen together with the aspirated capillary (FIG. 16b),
(C) A method (FIG. 16c) of loading a blood sample (specimen) self-collected using a puncture device as it is together with the blood collection site (16c),
(D) A method of absorbing the specimen on the filter paper piece (16d) or the cotton swab (16e) and loading the absorbed filter paper piece or the cotton swab together (FIGS. 16d and 16e),
Can be given. In addition, the sample collected by the dispenser (16a), the sample sucked by the capillary tube (16b), the sample collected by self-collection, etc. are absorbed together into one test piece (16f), and then the whole test piece is diluted with the dilution port (31). May be loaded (FIG. 17).

  The diluting part shown in FIG. 11b is particularly useful when the specimen loaded on the dilution port (31) is a specimen that has been absorbed in advance at the tip of a medium such as a cotton swab or a test piece (16f). The reason for this is that in the dilution section shown in FIG. 11b, the diluted solution discharged from the liquid feeding section is discharged from the side surface portion of the dilution port (31), so that the sample is absorbed by the swab or the test piece (16f) at the discharge position. This is because by combining the locations, the discharged diluted solution is directly sprayed on the specimen portion, and it can be expected to increase the dilution efficiency (FIG. 18).

  As the internal shape of the dilution port in the dilution apparatus of the present invention, an optimal shape may be selected according to the form of the specimen to be used. For example, when a droplet is used as a specimen, a dilution port (FIG. 19a) whose inner shape is cylindrical is preferable, and when a test piece is used as a specimen, a dilution port (FIG. 19b) whose inner shape is a rectangular parallelepiped is preferable. Other examples include a dilution port (FIG. 19c) having a conical or frustoconical internal shape. The volume of the dilution port may be appropriately determined in consideration of the target specimen dilution amount and dilution ratio.

  An example of the dilution part in the dilution apparatus of this invention is shown in FIG. The diluting device of the present invention can be produced by communicating the suction / discharge port (20) with one of the suction / discharge ports included in the suction / discharge means provided in the liquid feeding section. An exploded view of the dilution section shown in FIG. 20c is shown in FIG. A base (51b) having a dilution port and a base (51a) having a suction discharge port and a drain port (33) are fixed via a parallel pin (53) and a fixing screw (54), and a cover ( 52), a drain receiver is formed in the space between the cover (52) and the base (51b). When fixing the base (51a) and the base (51b), check valves (21a, 21b) for preventing backflow are inserted into the first flow path and the second flow path.

  In addition, although the liquid feeding part provided in the dilution apparatus shown in FIG. 3 is provided with two suction / discharge means, the liquid feeding part (FIG. 22) provided with one suction / discharge means is also shown in FIGS. The specimen can be diluted by a method similar to that shown in FIG. When one suction / discharge means is provided, (d) in the dilution step, when the suction / discharge means (18) is in the suction state (FIG. 22a), the volume of the diaphragm (41) of the means is provided in the dilution section. When the liquid level of the dilution port decreases and the suction / discharge means (18) is in the discharge state (FIG. 22b), the liquid level of the dilution port rises to the uppermost surface. For this reason, when an external factor such as an impact occurs in the dilution section when the suction / discharge means is in the discharge state, the diluted specimen on the top surface of the dilution port may spill out and the dilution performance may deteriorate. On the other hand, when two or more suction / discharge means are provided as in the liquid feeding section provided in the dilution apparatus shown in FIG. 3, when the suction / discharge means is in the suction state in the (d) dilution step, the diaphragm of the means has The liquid level of the dilution port provided in the diluting part is equal to the total volume (the total capacity of the diaphragm (41) of the suction / discharge means (18a, 18b in the case of the liquid feeding part provided in the diluting device shown in FIG. 3)). When the suction / discharge means is in the discharge state, the liquid level of the dilution port is equivalent to the capacity of one suction / discharge means (in the case of the liquid feed section provided in the dilution apparatus shown in FIG. 3, the suction / discharge means (18a) The liquid level rises to a position where it is reduced by the volume of the diaphragm (41) possessed by. In other words, since the liquid level is lower than the uppermost surface of the dilution port, even if some external factor such as an impact occurs in the dilution part, the sample dilution liquid does not spill out, and it is possible to exhibit more stable dilution performance. .

The dilution apparatus of the present invention is
A diluent supply unit for diluting the sample, a dilution unit for loading the sample and diluting the sample with the diluent, feeding the diluent and aspirating and discharging the diluted sample diluted by the dilution unit A liquid feeding section, the liquid feeding section having a suction port and a discharge port, a liquid feed means capable of feeding the diluted liquid from the suction port to the discharge port, and a diaphragm capable of holding the liquid And one or more suction / discharge means having a suction / discharge port and capable of sucking and discharging the liquid in the diaphragm,
It is characterized by having.

  In the diluting device of the present invention, the suction / discharge means provided in the liquid feeding section has a diaphragm capable of holding the liquid and a suction / discharge port, and is a means capable of sucking and discharging the liquid in the diaphragm. For example, the liquid can be sucked and discharged only by turning on / off the power supply to the electromagnetic component of the suction / discharge means. Therefore, the apparatus configuration can be greatly simplified, and the manufacturing cost can be greatly reduced. Furthermore, each means constituting the liquid feeding section can be concentrated on the manifold, thereby making it possible to greatly reduce the volume of the diluting device.

In addition, the dilution part with which the dilution apparatus of this invention is equipped,
A dilution port that holds the diluted solution or diluted sample and can load the sample,
A drain port capable of discharging the diluted solution or diluted sample, and
When the drain port is a dilution section provided on the outer peripheral portion of the dilution port, the apparatus can dilute the specimen not only in the liquid state but also in the specimen adsorbed on a filter paper piece or a cotton swab. Furthermore, the dilution unit provided in the dilution apparatus of the present invention,
A dilution port that holds the diluted solution or diluted sample and can load the sample,
A drain port provided on an outer peripheral portion of the dilution port and capable of discharging a diluted solution or a diluted specimen;
A first flow path for communicating the bottom of the dilution port with one of the suction and discharge ports of the suction and discharge means;
A second flow path for communicating the side surface portion of the dilution port with the first flow path;
When the sample is absorbed from a medium such as a filter paper piece, a cotton swab, or a test piece, the dilution unit has a check valve in each of the first channel and the second channel. Since the diluent discharged from the second flow channel is directly sprayed on the portion where the specimen is absorbed, more efficient specimen dilution is possible.

It is the figure which showed the flow-path system of the general liquid chromatograph apparatus. It is the schematic which showed an example of the dilution apparatus of this invention. After the dilution operation, a is a diagram showing a case where a diluted sample is introduced into the subsequent analysis means by a method, and b is a diagram showing a case where the diluted specimen is introduced into the subsequent analysis means by an automated introduction means. It is detail drawing which showed an example of the dilution apparatus of this invention. It is the figure which showed the basic operation | movement of the liquid feeding means and suction discharge means with which the dilution apparatus of FIG. 3 was equipped. It is the figure which showed the operation | movement in each process of the liquid feeding part with which the dilution apparatus of FIG. 3 was equipped. (A-1) and (a-2) show the operation in the cleaning process, and (b) show the operation in the discharge process. It is the figure which showed the operation | movement in each process of the liquid feeding part with which the dilution apparatus of FIG. 3 was equipped. (C) shows the operation in the specimen loading step, and (d-1) and (d-2) show the operation in the dilution step, respectively. FIG. 4 is a diagram showing the operation of each component (liquid feeding means, suction / discharge means, dilution section) when a specimen is diluted using the dilution apparatus shown in FIG. 3. In this figure, the symbols given to the respective steps correspond to the symbols in FIGS. It is the block diagram which showed another example of the dilution apparatus of this invention. a is a case where the liquid feeding means and the suction / discharge means are respectively arranged separately, b is a case where a motor-driven diaphragm pump or a piezoelectric element-driven diaphragm pump is used as the liquid feeding means, and c is a tube pump (peripheral) as the liquid feeding means. The configuration in the case of using a (talic pump) is shown. It is a figure which shows an example of the aspect which put together the liquid feeding part with which the dilution apparatus of this invention was equipped to one manifold. a is a general view and b is an exploded view. It is a figure which shows another example of the aspect which concentrated the liquid feeding part with which the dilution apparatus of this invention was equipped on one manifold. a is an overall view, and b and c are exploded views. It is the figure which showed the basic structure of the dilution part with which the dilution apparatus of this invention was equipped. a is a mode in which the flow channel when sucking the liquid and the flow channel when discharging is common, and b is a mode in which the flow channel when sucking the liquid and the flow channel when discharging the liquid are separated. Show. It is the schematic diagram which showed the principle of specimen dilution when using the dilution part shown in FIG. 11a. a is the operation during ejection, b is the operation during suction, and c is the operation during cleaning. FIG. 11B is a schematic diagram showing the progress of specimen dilution (preparation of diluted specimen) when using the dilution section shown in FIG. 11a. It is the schematic diagram which showed the principle of specimen dilution when using the dilution part shown in FIG. 11b. a is the operation during ejection, b is the operation during suction, and c is the operation during cleaning. FIG. 11B is a schematic diagram showing the progress of specimen dilution (preparation of diluted specimen) when the dilution section shown in FIG. 11B is used. It is the figure which showed the loading aspect of the test substance to the dilution port which the dilution part with which the dilution apparatus of this invention was equipped has. a is a mode of loading using a dispenser, b is a mode of loading using a capillary tube, c is a mode of loading a self-collected specimen for each blood collection site, and d is a mode of loading using a filter paper piece. , E each indicate a mode of loading using a cotton swab. It is the figure which showed the aspect loaded using the test substance absorbed to the test piece by the composite method among the test forms of the test substance to the dilution port which the dilution part with which the dilution apparatus of this invention was equipped has. It is the figure which showed the dilution aspect when the sample absorbed at the front-end | tip of a medium is diluted with the dilution part shown in FIG. 11b. It is the figure which showed the example of the shape of the dilution port which the dilution part with which the dilution apparatus of this invention was equipped has. It is a figure which shows an example of the dilution part with which the dilution apparatus of this invention was equipped. a is an overall view, b is a mode in which a flow path for sucking liquid and a flow path for discharging are made common, and c is a flow path for sucking liquid and a flow path for discharging. Each of the embodiments is shown. It is the figure which decomposed | disassembled the dilution part shown to FIG. 20c. It is the figure which showed another example of the liquid feeding part with which the dilution apparatus of this invention was equipped. It is the figure which showed the flow-path system of the dilution apparatus verified in the Example. It is the figure which showed the structure of the dilution part with which the dilution apparatus verified in the Example was equipped. It is the diagram which showed operation | movement of each component (a liquid feeding means, a suction discharge means, a dilution part) at the time of diluting a test substance using the dilution apparatus verified in the Example. In this figure, the symbols given to the respective steps correspond to the symbols in FIGS. It is the figure which showed the operation | movement in each process of the dilution apparatus verified in the Example. (A) shows the operation in the dilution port cleaning step, (b) shows the operation in the discharge step, and (c) shows the operation in the step of creating a gap for loading the specimen to the dilution port. It is the figure which showed the operation | movement in each process of the dilution apparatus verified in the Example. (D) shows the operation in the sample loading step, (e) shows the suction operation in the stirring / dilution step, and (f) shows the discharge operation in the stirring / dilution step. It is the figure which showed the change of the detector signal obtained in the Example. In a, the vertical axis represents the detector signal (mABU), and b represents the value obtained by converting the vertical axis into the dilution factor. In an Example, it is the figure which showed the change of the detector signal of each diluted specimen in the use method measured in order to calculate a dilution rate. FIG. 30 is a calibration curve created from the results of FIG. 29. FIG. It is the figure which showed the whole structure of the dilution apparatus used in order to create a calibration curve in the Example.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, these Examples do not limit this invention.

  In this embodiment, in order to confirm the dilution process, a detector (5) (UV-8020 manufactured by Tosoh Corporation) that can confirm a change in concentration between the liquid feeding section (15) and the dilution section (14) provided in the dilution apparatus. An ultraviolet-visible detector wavelength: 610 nm) was inserted for verification. FIG. 23 shows a flow path system of the diluting apparatus verified in this example. A 20% aqueous ethanol solution was used as the diluent (12), and 0.1% alizarin green (Wako Pure Chemical), which is a green pigment, was used as the sample (7).

  The liquid feeding section (15) in the diluting device of FIG. 23 has a suction port and a discharge port, and has a check valve (21a, 21b) capable of feeding the diluted solution from the suction port to the discharge port. (17) and a diaphragm (41) capable of holding liquid and two suction / discharge means (18a, 18b) having two suction / discharge ports and capable of sucking and discharging the liquid in the diaphragm (41). The Takasago Electric fixed discharge pump (MLP-200P), which is an electromagnetic diaphragm pump, is used as the liquid supply means (17), and the same Takasago Electric fixed discharge as the liquid supply means (17) as the suction discharge means (18a). The pump (MLP-200P) with the check valve removed is used as the suction discharge means (18b), and the Takasago Electric metered discharge pump (PKP-500) with the check valve removed is used. . In the above-described constant discharge pump, when there is a check valve, liquid is fed only in one direction by turning on / off the power to the electromagnetic coil, but when there is no check valve, as a simple suction discharge means Function.

  FIG. 24 shows the details of the diluting section (14) in the diluting apparatus verified in the present embodiment. The dilution part is a reciprocating operation system shown in FIG. 11a, and the dilution port (31) has a cylindrical shape, and has a diameter of 5 mmΦ × depth of 20 mm and a volume of about 400 μL.

FIG. 25 is a diagram showing the operation of the diluting apparatus verified in this example, and FIGS. 26 and 27 are diagrams schematically showing the operation in each step. A series of dilution operations will be described based on these.
(A) The suction / discharge means (18a) and the suction / discharge means (18b) are brought into the suction state (the diaphragm is inflated), and the power to the electromagnetic coil provided in the electromagnetic diaphragm pump as the liquid feeding means (17) is turned on. By switching / OFF at intervals of 0.3 seconds, the diluted solution is fed for about 1.3 minutes to clean the inside of the diluted port. At this time, the diluted solution exceeding the capacity of the dilution port (31) is discharged out of the system through the drain port (33) (FIGS. 25A and 26A).
(B) The suction / discharge means (18a) and the suction / discharge means (18b) are set to a discharge state (diaphragm is deflated). Accordingly, the total volume of the diaphragm (41) included in the suction / discharge means (18a) and the suction / discharge means (18b) is discharged from the dilution port (31), and the amount of liquid in the dilution port (31) is made constant. (FIG. 25 (b) and FIG. 26 (b)).
(C) The suction / discharge means (18a) and the suction / discharge means (18b) are set to a suction state (a state where the diaphragm is expanded). As a result, a gap corresponding to the total volume of the diaphragm (41) of the suction / discharge means (18a) and the suction / discharge means (18b) is created in the dilution port (31) (FIGS. 25C and 26C). )).
(D) With a gap formed in the dilution port (31), the sample (7) (0.1% alizarin green (manufactured by Wako Pure Chemical Industries) 20 μL) is dropped into the dilution port (31) (FIG. 25). (D) and FIG. 27 (d)).
(E) Of the suction and discharge means, only the suction and discharge means (18b) is repeatedly suctioned and discharged at intervals of 6 seconds to advance the dilution operation (FIGS. 25 (e) and (f) and FIGS. 27 (e) and (f) )).
(F) After the diluting operation for a predetermined time, the diluting liquid is sent by switching the power supply to the electromagnetic coil provided in the electromagnetic diaphragm pump as the liquid feeding means (17) at an interval of 0.3 seconds. The liquid is diluted and the inside of the dilution port is washed (FIG. 25 (a) and FIG. 26 (a)).

  The change of the detector signal when the above operation is performed is shown in FIG. 28a. After the sample is loaded, the absorbance increases immediately after the start of the stirring / dilution operation. After that, it can be seen that the concentration increases and decreases repeatedly in an attenuation curve and converges to a constant dilution concentration. FIG. 28b is a diagram when the vertical axis in FIG. 28a is converted into a change in dilution factor. For conversion to dilution ratio, first, the flow path before and after the detector (UV-visible detector) (5) is disconnected, and a syringe (10) is connected to one end of the detector, and 0.1% alizarin green ( 16 times, 32 times, 64 times, 128 times, 256 times or 512 times diluted solution (Wako Pure Chemical Industries) is introduced into the detector (5) using a syringe (FIG. 31), and the detector signal at each diluted solution (FIG. 29) and converted using a calibration curve (FIG. 30) prepared based on the signal. In addition, the horizontal axis of FIG. 30 is represented by the reciprocal (1 / D) of the dilution factor (D) (regression equation: y = 16643x).

  As can be seen from FIG. 28, as the suction / discharge operation of the suction / discharge means (18b) is repeated, the loaded sample draws an oscillating attenuation curve and dilutes to converge to a constant value. In the case of this example, it can be seen that the concentration converges to about 50 times (FIG. 28b).

  As described above, the diluting apparatus shown in FIG. 23, which is an example of the diluting apparatus of the present invention, has a simple configuration including one liquid feeding means and two suction / discharge means, and also controls the power supply to the electromagnetic coil. Therefore, the dilution device can be simplified and the cost can be reduced.

1: Liquid feed pump 2: Sample introduction valve 3: Analysis column 4: Column thermostat 5: Detector 6: Sample holding loop 7: Sample (specimen)
8: Eluent 9: Washing liquid 10: Syringe 11: Solenoid valve 12: Diluent 13: Diluted sample 14: Diluting part 15: Liquid feeding part 16a: Dispenser 16b: Capillary 16c: Blood collection site 16d: Filter paper piece 16e: Cotton swab 16f: Test piece 17: Liquid feeding means 18: Suction / discharge means 19: Suction port 20: Suction / discharge port 21: Check valve 22: Dilution unit holding base 31: Dilution port 32: Drain receiver 33: Drain port 34: Flow path 41: Diaphragm 42: Piston 43: Spring 44: Capacity adjustment screw 45: Electromagnetic coil 46: Power supply 51: Base 52: Cover 53: Parallel pin 54: Fixing screw 61: Manifold 62: Flow path

Claims (5)

A diluent supply section for diluting the specimen;
A dilution section for loading a specimen and diluting the specimen with the diluent;
A liquid feeding section capable of aspirating and discharging the diluted specimen and the diluted specimen diluted by the dilution section;
A diluting device comprising:
A liquid feeding means having a suction port and a discharge port, and capable of feeding the diluent from the suction port to the discharge port;
One or more suction / discharge means having a diaphragm capable of holding liquid and a suction / discharge port and capable of sucking / discharging the liquid in the diaphragm;
The dilution apparatus comprising: The liquid feeding part is
A liquid feeding means having a suction port and a discharge port, and capable of feeding the diluent from the suction port to the discharge port;
A first and second suction / discharge means having a diaphragm capable of holding liquid and two suction / discharge ports, and capable of sucking and discharging the liquid in the diaphragm;
With
The discharge port of the liquid feeding means communicates with one of the suction discharge ports of the first suction / discharge means,
The other of the suction and discharge ports of the first suction and discharge means communicates with one of the suction and discharge ports of the second suction and discharge means,
The other of the suction and discharge ports of the second suction and discharge means and the diluting portion can communicate with each other;
The dilution apparatus according to claim 1. The dilution section is
A dilution port that holds the diluted solution or diluted sample and can load the sample,
A drain port capable of discharging a diluted solution or a diluted specimen;
The dilution apparatus according to claim 1, wherein the drain port is provided on an outer peripheral portion of the dilution port. The dilution section is
A dilution port that holds the diluted solution or diluted sample and can load the sample,
A drain port provided on an outer peripheral portion of the dilution port and capable of discharging a diluted solution or a diluted specimen;
A first flow path for communicating the bottom of the dilution port with one of the suction and discharge ports of the suction and discharge means;
A second flow path for communicating the side surface portion of the dilution port with the first flow path;
The dilution apparatus according to claim 1, wherein a check valve is provided in each of the first flow path and the second flow path. The diluting device according to claim 1, wherein the liquid feeding means is a diaphragm pump.

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