JP2013040879A - Analyzer with analyte diluting device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analyzer with a diluting device, which is capable of handling analytes in various forms and has a simple configuration and is inexpensive.SOLUTION: The analyzer includes: solution delivery means 1 which delivers a solution; sample introducing means 2 which includes a loop 6 capable of being filled with a sample and sample filling means for filling the loop with the sample and can be switched between a state where the loop can be filled with the sample and a state where the sample filled in the loop can be delivered; and detection means 5. The sample filing means includes: a supply part which supplies a diluent for diluting the sample; a diluting part 14 which is loaded with the sample to dilute the sample with the diluent; and a solution delivery part 15 which includes diluent delivery means 17 having a suction port and a discharge port and capable of delivering the diluent from the suction port to the discharge port and one or more ducking/discharging means 18 including a diaphragm capable of holding a solution and a suction/discharge port and capable of sucking/discharging the solution in the diaphragm and is capable of delivering the diluent and of sucking/discharging a diluted sample diluted with the diluent.

Description

  The present invention relates to a sample analyzer equipped with a device for diluting a sample (specimen) to be introduced.

  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 to be introduced into an analyzer such as a liquid chromatograph device and methods for adding a hemolytic agent to a blood sample have been commercialized or disclosed. However, in any of the above-described methods, the sample (specimen) is in a liquid state accommodated in a blood collection tube, a sample cup, or the like, and it is difficult to handle the sample in other modes. 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.

  SUMMARY OF THE INVENTION An object of the present invention is to provide an analyzer equipped with a diluting device that can handle various types of specimens, has a simple structure, and is inexpensive.

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

That is, the first aspect of the present invention is:
A liquid feeding means for feeding the solution;
A loop that can be filled with a sample and a sample filling means that fills the loop with a sample are provided. The sample filling means can fill the loop with the sample and the sample filled in the loop is fed by the liquid feeding means. A sample introduction means capable of switching between a state in which liquid can be fed by the prepared solution;
An analysis device comprising a detection means,
The sample filling means comprises:
A diluent supply for diluting the sample;
A dilution section for loading the sample and diluting the sample with the diluent;
A dilution liquid feeding means having a suction port and a discharge port and capable of feeding the diluted liquid from the suction port to the discharge port, a diaphragm capable of holding the liquid, and a suction discharge port, and the liquid in the diaphragm One or more suction / discharge means capable of suction / discharge, and a liquid-feeding unit capable of sucking and discharging the diluted solution and the diluted sample diluted by the dilution unit,
It is the said apparatus which is a means provided with.

The second aspect of the present invention is as follows.
Liquid feeding means for feeding the first solution;
A loop that can be filled with a second solution and a first filling means that fills the loop with the second solution, a state in which the second solution can be filled into the loop by the first filling means, and the loop A first flow path switching means capable of switching between a state in which the second solution filled in the liquid can be fed by the first solution fed by the liquid feeding means;
A loop capable of filling the nth (n is 3 or more) solution and a (n-1) th filling means for filling the nth solution in the loop are provided, and the (n-1) th filling means The nth solution can be switched between a state in which the n solution can be filled in the loop and a state in which the nth solution filled in the loop can be fed by the first solution fed by the liquid feeding means. 1) the flow path switching means;
A flow path system provided in a loop provided with the (n-1) th flow path switching means in the (n-2) th flow path switching means,
A loop capable of filling a sample and a sample filling means for filling the loop with a sample are provided, and a state in which the sample can be filled by the sample filling means and a sample filled in the loop are fed by the liquid feeding means. A sample introduction means capable of switching between a state in which liquid can be fed by one solution;
Detection means;
The sample filling means comprises:
A diluent supply for diluting the sample;
A dilution section for loading the sample and diluting the sample with the diluent;
A dilution liquid feeding means having a suction port and a discharge port and capable of feeding the diluted liquid from the suction port to the discharge port, a diaphragm capable of holding the liquid, and a suction discharge port, and the liquid in the diaphragm One or more suction / discharge means capable of suction / discharge, and a liquid-feeding unit capable of sucking and discharging the diluted solution and the diluted sample diluted by the dilution unit,
It is the said apparatus which is a means provided with.

The third aspect of the present invention is as follows.
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 analyzer according to the first or second aspect, wherein a check valve is provided in each of the first channel and the second channel.

  A fourth aspect of the present invention is the analyzer according to any one of the first to third aspects, wherein the analyzer is a liquid chromatograph device.

  Hereinafter, the present invention will be described in detail.

A schematic view showing an example of the analyzer of the present invention is shown in FIG. The analyzer of the present invention shown in FIG.
A liquid feed pump (1) for feeding the eluent (8);
A loop (6) that can be filled with a sample and a sample filling means for filling the loop with a sample (specimen) are provided. A sample introduction means provided with a sample introduction valve (2) that can be switched between a state in which liquid can be fed by the eluent (8) fed by the liquid feed pump (1), and an analysis column (3) And a detector provided with a detector (5), the sample filling means,
A supply portion of a diluent (13) for diluting the sample (specimen);
A dilution section (14) for loading the sample and diluting the sample with the diluent (13);
A dilution liquid feeding means (17) having a suction port and a discharge port and capable of feeding the diluent from the suction port to the discharge port, a diaphragm capable of holding the liquid, and a suction discharge port. One or more suction / discharge means (18) capable of sucking and discharging the liquid, and feeding the diluted liquid (13) and feeding the diluted sample diluted by the dilution section (14) Part (15),
This is an analysis apparatus. In the present invention, the dilution liquid refers to a liquid added to a specimen in order to reduce the concentration of the target component and other components contained in the biological sample (specimen). In the case of blood, a liquid containing a hemolytic agent, which is added to a blood sample in order to separate and analyze a target component in blood cells, also corresponds to the diluent of the present invention.

  In the analyzer of the present invention, various types of flow analyzers can be constructed depending on what is provided on the outlet side of the sample introduction valve (2). For example, by providing separation means (for example, analysis column (3)) suitable for the specimen (sample) on the outlet side of the sample introduction valve (2), analysis of a liquid chromatograph device, a gas chromatograph device, a capillary electrophoresis device, etc. A device can be constructed. Moreover, a flow injection analyzer can be constructed by providing a pipe (22) as a reaction field on the outlet side of the sample introduction valve (2).

FIG. 3 shows an example of a diluting device (diluting part and liquid feeding part) in the sample filling means among the sample introducing means provided in the analyzer of the present invention. In the diluting apparatus shown in FIG.
A diluent feeding means (17) having a suction port and a discharge port and capable of feeding the diluent (13) 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 dilute solution 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).

As an example of the diluent feeding means (17) in the diluting apparatus shown in FIG. 3, 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) is given. It is done. 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 diluting liquid feeding means (17) is not limited to the electromagnetic diaphragm pump described above, but 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. If it is. As an example, a diaphragm pump that drives a piston (diaphragm) by a motor or a piezoelectric element, a tube pump represented by a peristaltic pump, and the like can be given.

As an example of the suction and discharge means (18a, 18b) in the dilution apparatus shown 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 dilution liquid feeding means (17) and one or more suction / discharge means (18) provided in the liquid feeding section (15) in the dilution apparatus shown in FIG.
(A) It may be arranged as individual means and each may be connected by external piping,
(B) A mode (FIGS. 5 and 6) 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 diluting apparatus shown in FIGS. 5 and 6, the dilute solution 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 dilution section (14) in the dilution apparatus shown in FIG. 3 is shown in FIG. 7a. 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. 7b shows another example of the dilution section (14) in the dilution apparatus shown in FIG. 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 (21b, 21a), 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 a simple cone or truncated cone shape as shown in FIG. 7b as long as the overflowed diluted solution flows smoothly in the direction of the drain groove as in the dilution portion shown in FIG. 7a. It may be any other shape.

  As the internal shape of the dilution port provided in the dilution section (14) in the dilution apparatus shown in FIG. 3, an optimal shape may be selected depending on the form of the specimen to be loaded. For example, when a droplet is used as a specimen, a dilution port (FIG. 8a) whose inner shape is cylindrical is preferable, and when a test piece is used as a specimen, a dilution port (FIG. 8b) whose inner shape is a rectangular parallelepiped is preferable. Another example is a dilution port (FIG. 8c) 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.

The aspect of the sample loaded on the dilution port (31) provided in the dilution section (14) in the analyzer of the present invention is not particularly limited. As an example,
(A) A method of directly loading a liquid sample using a dispenser such as a syringe or pipette,
(B) A method of aspirating a liquid specimen using a capillary and loading the specimen together with the aspirated capillary,
(C) A method of loading a blood sample (specimen) self-collected using a puncture device as it is with each blood collection site,
(D) A method of absorbing the specimen to the absorbent portion (16aa, 16ba) provided at the tip of the cotton swab (16a) or the test piece (16b) and loading the absorbed cotton swab (16a) or the test piece (16b) together (FIG. 9 to 12),
Can be given. Alternatively, a sample collected by a dispenser, a sample aspirated by a capillary, a sample collected by self-collection, and the like may be absorbed together into one test piece and then loaded together with the test piece to the dilution port.

  7B 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. The reason for this is that in the dilution section shown in FIG. 7b, the diluted liquid discharged from the liquid supply section is discharged from the side surface portion of the dilution port (31), so that the sample in the cotton swab or test piece (16b) is at the discharge position. This is because by combining the absorption part (16ba), the discharged diluted liquid is directly sprayed on the specimen absorption part (16ba), and it can be expected to increase the dilution efficiency (FIG. 13).

  In the analyzer shown in FIG. 2, the sample filling means is a means provided with the dilution part (14) and the liquid feeding part (15) shown in FIG. 3, and an analysis column (on the outlet side of the sample introduction valve (2)) The flow of dilution of the specimen (sample) and analysis of the diluted specimen in the embodiment provided with 3) will be described in detail with reference to FIGS.

(A) Dilution port cleaning process (FIG. 14 (a))
When the diluent (13) is sent by the diluent 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 diluting liquid feeding means (17) is driven (specifically, the power is periodically turned ON / OFF repeatedly), so that the diluting liquid is supplied to the diluting liquid feeding means (17) and the suction discharge. The liquid is fed in the order of the means (18a), the suction / discharge means (18b), and the diluting part (14), and when the diluting liquid overflows from the diluting part (14), the inside of the diluting part (14) and the liquid feeding part (15) And washing with a diluted solution. The sample introduction valve (2) is in a state in which the dilution part (14), the loop (6), and the liquid feeding part (15) are in communication with each other until the later described (d) dilution step (Load state). Then, the eluent (8) fed by the liquid feed pump (1) is directly introduced into the analysis column (3).

(B) Discharging process (FIG. 14 (b))
In a state where the diluting liquid feeding means (17) is stopped (that is, the diaphragm is deflated), the power to the electromagnetic coil provided in the suction / discharge means (18a, 18b) is turned off, the piston is moved downward, and the diaphragm (41 ) Is in a deflated state, the diaphragm (41) volume of diluent is discharged. Since the dilution liquid feeding means (17) side is closed (OFF state) by the check valve on the discharge port side of the dilution liquid feeding means (17), the dilution liquid only on the dilution section (14) side. Is discharged.

(C) Specimen loading process (FIG. 15 (c))
After the sample (7) is loaded in the diluting section (14), the power to the electromagnetic coil provided in the suction / discharge means (18a, 18b) is turned on while the dilute solution feeding means (17) is stopped and the piston is turned on. Is moved upward to bring the diaphragm (41) into a swelled state, and the diluted solution (diluted sample) containing the sample is aspirated into the diaphragm (41). Note that the non-return valve on the discharge port side of the dilute solution feeding means (17) closes the dilute solution feed means (17) side, so that the diaphragm (41) of the suction and discharge means (18a, 18b) is provided. Diluted specimen is aspirated only in

(D) Dilution step (FIG. 15 (d))
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.

(E) Diluted sample introduction step (FIG. 16 (e))
After completion of the sample dilution, the sample introduction valve (2) is switched from the Load state to the state where the eluent (8) -loop (6) -analysis column (3) communicates (Injection state). The diluted sample packed in the loop (6) is introduced into the analysis column (3) in the form of being pushed out by the eluent (8), and the elution and analysis of the sample components by the eluent are performed.

  When the specimen (sample) is diluted and analyzed using the analyzer shown in FIGS. 14 to 16, the suction and discharge states of the diluent feeding means (17) and the suction and discharge means (18a and 18b) are provided in the dilution section. A diagram summarizing the liquid level position of the dilution port (31) and the switching position of the sample introduction valve (2) is shown in FIG. In FIG. 17, the symbols given to the respective steps correspond to the symbols in FIGS.

Another example of the analyzer of the present invention is shown in FIG. The analyzer shown in FIG.
A feed pump (1a) for feeding an eluent A (buffer A: also serving as an extrusion solution) (8a), and a first for introducing the eluent B (buffer B) (8b) into the analytical column (3). A flow path switching valve (12a), a second flow path switching valve (12b) for introducing the eluent C (buffer C) (8c) into the analysis column (3), and a loop capable of filling the buffer B (6b) and a loop (6c) that can be filled with the buffer C, and the second flow path switching valve (12b) is provided in a loop (6b) provided in the first flow path switching valve (12a). And the flow path system,
A sample introduction means having a sample filling means and a sample introduction valve (2) similar to the analyzer shown in FIGS. 14 to 16 provided on the outlet side of the first flow path switching valve (12a);
It is an apparatus provided with an analytical column (3) and a detector (5). As the first channel switching valve (12a), the second channel switching valve (12b), and the sample introduction valve (2), a two-position switching six-way valve that is usually used in a liquid chromatograph can be used.

  The flow of sample dilution and sample analysis in the analyzer shown in FIG. 27 will be described in detail with reference to FIGS.

From (a) dilution port washing step (FIG. 28) to (e) dilution stirring step (FIG. 32), the specimen (sample) is diluted by the same method as in FIGS. 14 (a) to 15 (d). The sample introduction valve (2) is in a state (Load state) in which the dilution part (14) -the loop (6a) -the liquid feeding part (15) communicate with each other. In the Load state, the liquid feeding pump (1a) The buffer A (8a) to be fed is directly introduced into the analysis column (3). The first flow path switching valve (12a) is in a state (OFF state) in which the loop (6b) can be filled with the buffer B (8b), and the second flow path switching valve (12b) is buffered in the loop (6c). Let C (8c) be in a state where it can be filled (OFF state).

(F) Diluted sample introduction step (FIG. 33)
After completion of the sample dilution, the sample introduction valve (2) is switched from the Load state to the state where the eluent (8) -loop (6a) -analysis column (3) communicates (Injection state). The diluted specimen filled in the loop (6a) is introduced into the analysis column (3) in the form of being pushed out by the buffer A (extrusion liquid) (8a), and the sample components are eluted and analyzed by the buffer A (8a).

(G) Step gradient analysis process (FIG. 34)
After the diluted sample introduction is completed, the sample introduction valve (2) is returned from the injection state to the load state, so that the sample (sample) can be diluted again. On the other hand, the first flow path switching valve (12a) is in a state (ON state) in which the flow path for sending the buffer A (8a) and the loop (6b) filled with the buffer B are connected to each other. The switching valve (12b) switches to a state (ON state) in which the loop (6b) filled with the buffer B and the loop (6c) filled with the buffer C are connected. Thus, the buffer B (extruded solution) is introduced into the analysis column (3) in the order of buffer B and buffer C, and the sample components are eluted by the buffer B, and subsequently the sample components are eluted and analyzed by the buffer C. .

  The aspiration / discharge state (ON / OFF state) of the dilute solution feeding means (17) and the suction / discharge means (18a, 18b) when the specimen (sample) is diluted and analyzed using the analyzer shown in FIG. FIG. 35 shows a diagram summarizing the liquid level position of the dilution port (31) provided in the dilution section and the switching positions of the sample introduction valve (2) and the flow path switching valves (12a, 12b). In FIG. 35, symbols given to the respective steps correspond to the symbols in FIGS.

  In the analyzer shown in FIG. 27, there are three types of eluents (buffers) to be sent to the analysis column, and the analyzer is provided with two flow path switching means. The type of the liquid (buffer) is not limited. When the number of eluents (buffers) sent to the analysis column is n, (n-1) channel switching means may be provided.

  In the analyzer shown in FIG. 27, switching in the high pressure state (that is, pressure fluctuation) is always required only twice, so that no extra noise is given to the detector provided in the analyzer, and more accurate analysis is possible. Therefore, it is preferable. Note that the sample introduction means may be provided in a loop provided in the first flow path switching means. In that case, since switching in a high pressure state (ie, pressure fluctuation) is always required only once, more accurate analysis is possible. This is expected to be possible (Japanese Patent Application No. 2010-268560).

  14 to 16 and FIG. 27, the liquid feeding section provided in the dilution apparatus of the analyzer includes two suction / discharge means. However, even the liquid feeding section provided with one suction / discharge means may be To 15 and the method shown in FIGS. 27 to 32 can be used to dilute the specimen. When one suction / discharge means is provided, when the suction / discharge means is in the suction state in the dilution process, the liquid level of the dilution port provided in the dilution section is reduced by the volume of the diaphragm of the means, and the suction / discharge means Is in the discharge state, 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, when the suction / discharge means is in the suction state in the dilution (stirring) step, the total capacity of the diaphragm of the means (analyzer shown in FIGS. 14 to 16 and FIG. 27) In the case of the liquid feeding section provided in the dilution device, the liquid level of the dilution port provided in the dilution section is lowered by the amount of the diaphragm (41) of the suction / discharge means (18a, 18b), and the suction / discharge means When in the discharge state, the liquid level of the dilution port has a capacity corresponding to one suction discharge means (in the case of the liquid feed section provided in the dilution apparatus of the analyzer shown in FIGS. 14 to 16 and 27, the suction discharge means (18a The liquid level rises to a position lowered by the diaphragm (41) of the diaphragm (41). 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 analyzer of the present invention comprises a liquid feeding means for feeding a solution, a loop capable of filling a sample, and a sample filling means for filling the loop with a sample, and the sample filling means can fill the loop with the sample. A sample introduction means capable of switching between a state and a state in which the sample filled in the loop can be fed by the solution fed by the liquid feeding means, and a detection means, and the sample filling means comprises a sample A diluent supply section for diluting the sample, a dilution section for loading the sample and diluting the sample with the diluent, and having a suction port and a discharge port, and the diluent can be fed from the suction port to the discharge port The diluting liquid feeding means and the diluting liquid feeding means, the diaphragm capable of holding the liquid, and one or more suction and discharging means capable of sucking and discharging the liquid in the diaphragm are provided. Dilution diluted by part It is characterized in that suction and discharge of the charges and a, and a liquid supply unit as possible.

  In the sample filling means provided in the analyzer 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 can suck and discharge the liquid in the diaphragm. By using the means, for example, the liquid can be sucked and discharged only by the power ON / OFF operation to the electromagnetic parts included in the suction and discharge means. Therefore, the apparatus configuration can be greatly simplified, and the manufacturing cost can be greatly reduced. Further, each means constituting the liquid feeding section can be concentrated on the manifold, and this makes it possible to greatly reduce the volume of the sample filling means in the analyzer.

In addition, the dilution part in the sample filling means provided in the analyzer 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. Therefore, as a result, the analysis accuracy of the sample component by the analyzer can be improved. Since the dilution section circulates the diluted solution and the sample (specimen), it is possible to efficiently dilute the sample even when the sample load is large.

It is the figure which showed the flow-path system of the general liquid chromatograph apparatus. It is the schematic diagram which showed an example of the analyzer of this invention. It is the figure which showed an example of the dilution apparatus with which the analyzer of this invention was equipped. It is the figure which showed the basic operation | movement of the liquid feeding means and the suction discharge means with which the dilution apparatus shown in FIG. 3 was equipped. It is a figure which shows an example of the aspect which concentrated the liquid feeding part on one manifold among the dilution apparatuses with which the dilution apparatus shown in FIG. 3 was equipped. 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 on one manifold among the dilution apparatuses with which the dilution apparatus shown in FIG. 3 was equipped. 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 among the dilution apparatuses with which the dilution apparatus shown in FIG. 3 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 figure which showed the example of the shape of the dilution port which a dilution part has among the dilution apparatuses with which the dilution apparatus shown in FIG. 3 was equipped. It is the figure which showed an example of the cotton swab used for the load of the sample analyzed with the analyzer of this invention. It is the figure which showed an example of the sample load aspect to the sample introduction means with which the analysis apparatus of this invention was used using the cotton swab shown in FIG. It is the figure which showed an example of the test piece used for the load of the sample analyzed with the analyzer of this invention. It is the figure which showed an example of the sample load aspect to the sample introduction means with which the test piece shown in FIG. 11 was equipped with the analyzer of this invention. It is the figure which showed the dilution aspect when the test substance absorbed in the test piece shown in FIG. 11 is diluted with the dilution part shown in FIG. 7b. It is the figure which showed the operation | movement of each process in an example of the analyzer of this invention. (A) shows the operation in the dilution port cleaning process, and (b) shows the operation in the discharge process. It is the figure which showed the operation | movement of each process in an example of the analyzer of this invention. (C) shows the operation in the specimen loading process, and (d) shows the operation in the dilution process. It is the figure which showed the operation | movement of the (e) dilution sample introduction | transduction process in an example of the analyzer of this invention. 6 is a diagram showing the operation of each component (liquid feeding means, suction / discharge means, dilution section, sample introduction means) when a sample is analyzed using an example of the analyzer of the present invention. In this figure, the symbols given to the respective steps are the same as those in FIGS. 2 is a calibration curve used for calculating the dilution rate in Example 1. FIG. 2 is a chromatogram showing a dilution process when the sample load is fixed to 10 μL in Example 1 and the sample concentration is changed. The horizontal axis indicates the number of measurements (# 1 to # 13), and the vertical axis indicates the output of the detector. It is a figure which shows the result of having plotted the peak area in each sample density | concentration (13th measurement, # 13 of FIG. 19a to 19e) based on FIG. 2 is a chromatogram showing a dilution process when the sample concentration is fixed to 50 mg / 50 mL and the sample load is changed in Example 1. FIG. The horizontal axis indicates the number of measurements, and the vertical axis indicates the output of the detector. It is a figure which shows the result of having plotted the peak area in each sample load amount (13th measurement, # 13 of FIGS. 21a to 21f) based on FIG. In Example 1, it is the result which put together the chromatogram for every measurement (# 1 to # 13). In each chromatogram, the horizontal axis represents the elution time, the vertical axis represents the detector output, and the number shown at the top represents the total number of suction / discharge times in the dilution operation. It is a figure which shows the result of having confirmed reproducibility in Example 1. FIG. The vertical axis represents the peak area at the 13th measurement (# 13), and the horizontal axis represents the number of executions (n). In the analyzer of Example 2, it is the chromatogram which showed the process of dilution when fixing a sample concentration to 50 mg / 50mL and changing a sample load. The horizontal axis indicates the number of measurements (# 1 to # 13), and the vertical axis indicates the output of the detector. It is a figure which shows the result of having plotted the peak area (13th measurement, # 13 of FIG. 25a to 25g) in each specimen (sample) load amount based on FIG. It is the figure which showed another example of the analyzer of this invention. It is the figure which showed the operation | movement in the (a) dilution port washing | cleaning process of the analyzer shown in FIG. It is the figure which showed the operation | movement in the (b) dilution liquid discharge process (metering process) of the analyzer shown in FIG. It is the figure which showed the operation | movement in the (c) sample load clearance preparation process of the analyzer shown in FIG. It is the figure which showed the operation | movement in the (d) sample loading process of the analyzer shown in FIG. It is the figure which showed the operation | movement in the (e) dilution stirring process of the analyzer shown in FIG. It is the figure which showed the operation | movement in the (f) dilution sample introduction process of the analyzer shown in FIG. It is the figure which showed the operation | movement in the (g) step gradient analysis process of the analyzer shown in FIG. FIG. 28 is a diagram showing the operation of each component (liquid feeding means, suction / discharge means, dilution section, sample introduction means, flow path switching means) when a sample is analyzed using the analyzer shown in FIG. 27. In this figure, the symbols given to the respective steps are the same as those in FIGS. It is the figure which showed the change of the chromatogram with respect to the load amount at the time of changing sample load amount in Example 3. FIG. The horizontal axis represents the measurement time, and the vertical axis represents the detector output. It is a figure which shows the result of having plotted the peak area value of (a) A1c or (b) A0 in each sample load based on FIG. It is the figure which showed the change of the chromatogram with respect to a dilution rate at the time of changing a sample dilution rate in Example 3. FIG. The horizontal axis represents the measurement time, and the vertical axis represents the detector output. It is a figure which shows the result of having plotted the peak area value of (a) A1c or (b) A0 in each sample dilution rate based on FIG. In the analyzer shown in FIG. 27, it is a figure which shows the result of having evaluated the reproducibility at the time of loading a liquid sample directly. (A) is a representative chromatogram, (b) is the transition of retention time (RT) of A1c and A0 in each measurement, (c) is the transition of A1c (%) in each measurement, (d) is It is a calibration curve used for calculating the dilution rate. In the analyzer shown in FIG. 27, it is a figure which shows the result of having evaluated the reproducibility when it loaded in the aspect which made the absorbent body of the cotton swab absorb the sample liquid. (A) is a representative chromatogram, (b) is the transition of retention time (RT) of A1c and A0 in each measurement, (c) is the transition of A1c (%) in each measurement, (d) is It is a calibration curve used for calculating the dilution rate. It is a figure which shows the result of having evaluated the reproducibility when it loads with the aspect which made the absorber of a test piece absorb the sample liquid with the analyzer of Example 4. FIG. (A) is a representative chromatogram, (b) is the transition of retention time (RT) of A1c and A0 in each measurement, (c) is the transition of A1c (%) in each measurement, (d) is It is a calibration curve used for calculating the dilution rate.

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

Example 1
Using the analyzer of the present invention shown in FIGS. 14 to 16, the usefulness of the analyzer of the present invention was evaluated.

  The liquid feed pump (1) is Tosoh DP-8020, the sample introduction valve (2) is a 2-position switching valve, the loop (6) is a 0.5 μL capacity loop, and the analytical column (3) is Tosoh TSKgel. ODS-100V (inner diameter 1 mm ID, length 35 mm, particle size 3 μm), detector (5) UV-visible detector UV-8020 (microcell) (detection wavelength: 550 nm) manufactured by Tosoh, eluent (buffer) ( 8) and dilute solution (13) were 30% acetonitrile aqueous solutions, respectively, and alizarin green (manufactured by Wako Pure Chemical Industries), which is a green pigment, was used as a sample (specimen).

  The diluting section (14) employs a format (reciprocating operation method) in which the suction / discharge flow paths shown in FIG. The dilution port (31) has a cylindrical shape (FIG. 8a) with a diameter of 5 mmΦ × depth of 20 mm and a volume of about 400 μL. The diluent feeding means (17) is a Takasago Electric fixed discharge pump MVP-50, which is an electromagnetic diaphragm pump, and the suction discharge means (18a) is the same pump as the diluent feeding means (17) except for the check valve. As the suction discharge means (18b), those obtained by removing the check valve from the Takasago Electric fixed discharge pump PKP-500 were used.

Dilution of the sample and analysis of the diluted sample were performed by the following method.
(A) With the diaphragm (41) of the suction / discharge means (18a, 18b) swelled (ON state), the ON / OFF state of the dilute solution feeding means (17) is changed for about 0.3 minutes at intervals of 3 seconds. By switching, the dilution liquid is fed and the inside of the dilution port (31) is washed (FIG. 14 (a)).
(B) The diaphragm (41) of the suction / discharge means (18a, 18b) is in a deflated state (OFF state), and the diluent is discharged, whereby the volume of the diluent in the dilution port (31) is made constant (FIG. 14 (b)).
(C) The suction / discharge means (18a, 18b) is turned on to suck the diluent, thereby creating a gap of a certain volume in the dilution port (31) and loading the sample (FIG. 15 (c)). .
(D) The sample is diluted by reciprocating the ON / OFF state of the suction / discharge means (18b) five times at intervals of 2.4 seconds (FIG. 15 (d)).
(E) The sample introduction valve (2) is switched from the Load state to the Injection state, and the diluted sample in the loop (6) is introduced into the analysis column (3) by pushing it out with the eluent (8) (FIG. 16 (e) )).
(F) After waiting for about 15 seconds (while switching the sample introduction valve (2) from the injection state to the load state), the operations from (a) to (e) were performed again, and the diluted sample was introduced 13 times in total.

  A calibration curve used for calculating the dilution rate is shown in FIG. FIG. 18 shows an analytical column obtained by diluting a sample (alizarin green) 1-fold (50 mg / 50 mL (1 mg / mL)), 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, and 200-fold using the procedure described above. It is created on the basis of the result obtained by introducing in (3) (FIG. 18a is represented by an integer axis, and FIG.

  In the sample analysis using the analyzer of the present invention shown in FIGS. 14 to 16, the chromatogram showing the tendency of the peak area (height) when the sample load is fixed at 10 μL and the sample concentration is changed is shown. 19 shows. The concentration of the loaded sample (alizanin green) is 25 mg / 50 mL (a), 50 mg / 50 mL (b), 75 mg / 50 mL (c), 100 mg / 50 mL (d), 125 mg / 50 mL (e). It can be seen that a constant peak height, that is, a constant dilution rate is exhibited from the fourth to fifth sample dilutions when any concentration of the sample solution is used. When the fourth to fifth sample dilution is converted into the number of reciprocations in the ON / OFF state in the suction / discharge means (18b), it corresponds to 20 to 25 reciprocations.

  FIG. 20 shows the relationship between the concentration of the loaded sample solution and the peak area at the 13th sample dilution (# 13 in FIGS. 19a to 19e). As can be seen from FIG. 20, when the sample loading is constant, a good linear relationship is recognized between the sample concentration and the peak area, and quantitative dilution is performed.

  In sample analysis using the analyzer of the present invention shown in FIGS. 14 to 16, the peak area (height) tendency when the sample concentration is fixed to 50 mg / 50 mL (1 mg / mL) and the sample loading is changed. A chromatogram showing the above is shown in FIG. The amount of the sample (alizanine green) solution loaded is 2.5 μL (a), 5 μL (b), 10 μL (c), 15 μL (d), 20 μL (e), and 25 μL (f). It can be seen that, when any sample amount is used, a constant peak height, that is, a constant dilution rate is exhibited from the fourth to fifth sample dilutions. When the fourth to fifth sample dilution is converted into the number of reciprocations in the ON / OFF state in the suction / discharge means (18b), it corresponds to 20 to 25 reciprocations.

  FIG. 22 shows the relationship between the sample load and the peak area at the 13th sample dilution (# 13 in FIGS. 21a to 21f). From FIG. 22, when the sample concentration is constant, in the range of 2.5 μL to 15 μL (FIGS. 21a to 21d), a good linear relationship is recognized between the sample load and the peak area, and quantitative dilution is performed. However, it became a tendency to deviate from the linear relationship (the tendency for the graph to lie down) at loads higher than that. The reason for this is that, in the analyzer of the present invention shown in FIGS. 14 to 16, the sample is diluted and stirred by reciprocating the diaphragm volume of the suction and discharge means (18b), so that the convection is insufficient and the sample load amount is low. This is considered to be because a sample having a stock solution concentration far from the interface between the sample and the diluent tends to be difficult to be diluted.

  The results of confirming the reproducibility of the analyzer of the present invention shown in FIGS. 14 to 16 are shown in FIGS. The amount of the loaded sample solution is 10 μL (using a microsyringe), the concentration is 50 mg / 50 mL (dilution rate 1), and the sample dilution and the analysis of the diluted sample are performed 13 times. FIG. To # 13) is an example of a chromatogram. In FIG. 23, the number of times described in the upper part of each chromatogram is the total number of reciprocations in the ON / OFF state of the suction / discharge means (18b). It can be seen from the fourth to fifth sample dilutions (# 4 to # 5) that a constant peak height, i.e. a constant dilution rate, is exhibited. The fourth to fifth sample dilution corresponds to 20 to 25 reciprocations when converted to the number of reciprocations in the ON / OFF state in the suction / discharge means (18b), and 1.6 to 2.0 minutes when converted to time.

  FIG. 24 and Table 1 show the results of summarizing the peak area (height) and the dilution rate for each measurement cycle (n) at the 13th sample dilution (# 13 in FIG. 23). Under this condition, about 100-fold dilution is performed, CV = about 5.3% due to fluctuation of peak height, CV = about 3.8% when the dilution rate is converted from the area and area, and the reciprocal of the dilution rate. It can be seen that the analysis apparatus of the present invention shown in FIGS. 14 to 16 can stably dilute and analyze the sample.

実施例２
図１４から１６に示す本発明の分析装置のうち、希釈部（１４）を図７ｂに示す吸引／吐出の流路が異なる形式（循環動作方式）とし、希釈ポート（３１）を２ｍｍ×５ｍｍ×深さ２０ｍｍの直方体形状（図８ｂ）とした分析装置（以下、実施例２の分析装置とする）を用いて、実施例１と同様に、試料の希釈および希釈試料の分析を行なった。

Example 2
14 to 16, the diluting section (14) is of a type (circulation operation system) having a different suction / discharge flow path shown in FIG. 7b, and the dilution port (31) is 2 mm × 5 mm × The sample was diluted and the diluted sample was analyzed in the same manner as in Example 1 using an analyzer (hereinafter referred to as the analyzer of Example 2) having a rectangular parallelepiped shape (FIG. 8 b) having a depth of 20 mm.

  FIG. 25 shows a chromatogram showing the tendency of the peak area (height) when the sample concentration is fixed to 50 mg / 50 mL and the sample load is changed in the sample analysis using the analyzer of Example 2. . The amount of sample (alizanine green) solution loaded was 2.5 μL (a), 5 μL (b), 10 μL (c), 15 μL (d), 20 μL (e), 25 μL (f), 50 μL (g) It is. It can be seen that, when any sample amount is used, a constant peak height, that is, a constant dilution rate is exhibited from the fourth to fifth sample dilutions. When the fourth to fifth sample dilution is converted into the number of reciprocations in the ON / OFF state in the suction / discharge means (18b), it corresponds to 20 to 25 reciprocations.

  FIG. 26 shows the relationship between the sample load and the peak area at the 13th sample dilution (# 13 in FIGS. 25a to 25g). A good linear relationship is recognized in a wide range of 2.5 μL to 50 μL, and it is understood that quantitative dilution is performed. As described above, in the analyzer of the present invention shown in FIGS. 14 to 16 adopting the reciprocating operation method (FIG. 7a) in the dilution section (14), the linear relationship was obtained up to 15 μL. It turns out that the capacity | capacitance from which the linear relationship is acquired with the analyzer of Example 2 has expanded 2 times or more. Therefore, since the analyzer of Example 2 adopting the circulation operation method (FIG. 7b) in the dilution section (14) can efficiently perform the stirring / dilution operation, the sample components can be accurately obtained even when the sample load is large. Can be analyzed.

Example 3
The amount of glycohemoglobin in blood components was analyzed using the analyzer of the present invention (FIG. 27) using the step gradient channel system disclosed in Japanese Patent Application No. 2010-268560.

  The liquid feed pumps (1a, 1b, 1c) are UP002SZWB made by UNIFLOWS, the sample introduction valve (2) and the flow path switching valves (12a, 12b) are a first stator and a first stator provided with at least two through holes. The second stator having the same number of through holes as the first stator, the first stator, and the second stator are liquid tightly sandwiched, and the outer periphery that allows the through holes provided in the first stator and the second stator to communicate with each other Rotor comprising: a flow path switching valve provided with a rotor seal provided with a gear on the part; and a means for transmitting the power from the drive means by meshing with a drive means and a gear provided on the outer periphery of the rotor seal A valve (Japanese Patent Application No. 2010-031095) having a seal rotation means is provided as a loop (6a) on the rotor seal of the sample introduction valve (2). The loop (6b) is a loop with a capacity of 11 μL provided outside the flow path switching valve (12a), and the loop (6c) is a loop with a capacity of 47 μL provided outside the flow path switching valve (12b). ) Is Tosoh TSKgel SP-NPR (inner diameter 0.3 mm ID, length 35 mm, particle size 3 μm), detector (5) is Tosoh UV-Vis detector UV-8020 (microcell) (detection wavelength: 415 nm) The eluents (buffers) (8a, 8b, 8c) were prepared by changing the concentration of the eluent for the glycohemoglobin analyzer HLC-723GHbVIII manufactured by Tosoh Co., Ltd. Level 1 or 2) or a Tosoh HbA1c calibrator (CALIB_1 or CALIB_2) diluted with pure water It was used.

  The diluting section (14) employs a form (circulation operation method) in which the suction / discharge flow paths shown in FIG. 7b are different. The dilution port (31) has a rectangular parallelepiped shape (FIG. 8b) of 2 mm × 5 mm × depth 20 mm. The diluent feeding means (17) is a Takasago Electric fixed discharge pump MVP-50, which is an electromagnetic diaphragm pump, and the suction discharge means (18a) is the same pump as the diluent feeding means (17) except for the check valve. As the suction discharge means (18b), those obtained by removing the check valve from the Takasago Electric fixed discharge pump PKP-500 were used.

Dilution of the sample and analysis of the diluted sample were performed by the following method.
(I) The sample was loaded and diluted in the same manner as in Examples 1 (a) to (d) (FIGS. 28 to 32). The form of the added sample is
(I-1) A form in which a liquid sample is directly loaded,
(I-2) Aspure cotton swab AP-8 (manufactured by ASONE, product number: 1-8584-) consisting of a core rod portion of about 0.5 mmφ (aluminum material) and an absorbent portion (16aa) of about 1 mmφ wound with absorbent cotton. 08) A form (FIGS. 9 and 10) in which the excess liquid droplets are removed by immersing the sample liquid in the absorption part (16aa) of (16a),
(I-3) An absorption part (16ba) made of cellulose filter paper having a width of 2.5 mm and a width of 2.5 mm was heat-sealed to the tip of a resin sheet having a width of 2.5 mm, a length of 200 mm, and a thickness of 200 μm. A form (FIGS. 11 and 12) in which excess liquid droplets are removed by immersing the specimen (16b) in the sample liquid
One of them.
(II) After completion of sample dilution, the sample introduction valve (2) is switched from the Load state to the Injection state, and the diluted sample filled in the loop (6a) is pushed out by the buffer A (extrusion solution) (8a) (11 μL per minute) ) Into the analytical column (3) (FIG. 33).
(III) 0.25 minutes after the start of introduction of the buffer A (extrusion liquid) (8a) into the analytical column (3), the sample introduction valve (2) is returned from the injection state to the load state, and the specimen (sample) is diluted again. Is possible. On the other hand, the two flow path switching valves (12a, 12b) are respectively switched from the OFF state to the ON state, so that the buffer B and the buffer C filled in the loop (6b, 6c) are pushed out by the buffer A to the analysis column. To be introduced. As a result, a step gradient is performed in which the buffer A is sent from 0 to 0.25 minutes after the start of introduction, the buffer B is sent from 0.25 to 1.25 minutes, and the buffer C is sent from 1.25 minutes onwards. FIG. 34).

  In the sample analysis using the analyzer of the present invention shown in FIG. 27, a sample obtained by dissolving a freeze-dried product of HbA1c calibrator (CALIB_2) in 0.5 mL of pure water was directly added to 5 μL (a), 10 μL (b). FIG. 36 shows chromatograms when 15 μL (c), 20 μL (d), and 40 μL (e) are loaded. FIG. 37 shows the result of plotting the peak area values of A1c and A0 against the sample load based on the result of FIG. It can be seen that the areas of A1c and A0 increase in proportion to the amount of sample loaded, and the correlation is good.

  In the sample analysis using the analyzer of the present invention shown in FIG. 27, a sample (stock solution) (a) obtained by dissolving a freeze-dried product of HbA1c calibrator (CALIB_2) in 0.25 mL of pure water and 2 according to the method used. FIG. 38 shows a chromatogram when 10 μL of a sample diluted twice (b), 4 times (c), and 8 times (d) is directly loaded. FIG. 39 shows the result of plotting the peak area values of A1c and A0 against the concentration (dilution rate) of the loaded sample solution based on the results of FIG. It can be seen that the areas of A1c and A0 increase in proportion to the loaded sample concentration (dilution rate), and the correlation is also good.

  FIG. 40 and Table 2 show the reproducibility evaluation results when the liquid sample was directly loaded using the analyzer of the present invention shown in FIG. A sample prepared by dissolving a freeze-dried product of HbA1c control (level 2) manufactured by Tosoh in 0.5 mL of pure water was used. FIG. 40a is a representative chromatogram, FIG. 40b is a result of plotting the elution time of A1c and A0 for each measurement, FIG. 40c is a result of plotting A1c (%) for each measurement, and FIG. 40d is for calculating the dilution rate. Table 2 summarizes the elution time, peak area, and A1c (%) of A1c and A0 for each measurement. The reproducibility (CV) of elution time of A1c and A0 is 0.51% and 0.47%, respectively, and even if the sample is introduced through a diluting device, a phenomenon such as sample blanking occurs. Suggests not. The area reproducibility (CV) of A1c and A0 was about 7.2% and 7.3%, respectively, but the reproducibility (CV) of A1c (%) was very good at 0.52%. It was confirmed that there was no problem in quantitativeness. It should be noted that the dilution rate obtained under these conditions was calculated to be 1 / 45.5 from the calibration curve (FIG. 40d) prepared in advance by the usage method.

液体試料の代わりに、綿棒（図９）先端の吸収部に試料を吸収させた形態で負荷したときの、再現性評価結果を図４１および表３に示す。試料は東ソー製ＨｂＡ１ｃコントロール（レベル２）凍結乾燥品を純水０．２５ｍＬに溶解したものを使用した。図４１ａは代表的なクロマトグラム、図４１ｂは測定毎のＡ１ｃおよびＡ０の溶出時間をプロットした結果、図４１ｃは測定毎のＡ１ｃ（％）をプロットした結果、図４１ｄは希釈率を算出するために用いた検量線、表３は測定毎のＡ１ｃおよびＡ０の溶出時間ならびにピーク面積、Ａ１ｃ（％）をまとめたものである。綿棒を媒体として試料を負荷および希釈操作を行ない、その希釈検体を液体クロマトグラフに導入、分析を行なっても良好なクロマトグラムが得られていることがわかる（図４１ａ）。Ａ１ｃおよびＡ０の溶出時間の再現性（ＣＶ）はそれぞれ、０．７１％、０．４４％と良好であり、希釈装置を介して注入を行なっても、試料の空打ちなどの現象が生じていないことを示唆している。Ａ１ｃおよびＡ０のピーク面積の再現性（ＣＶ）はそれぞれ、１３．５％、１３．５％程度と、前記の液体試料を直接負荷する方式より劣っていたものの、Ａ１ｃ（％）の再現性（ＣＶ）は０．１７％と非常に良好であり、定量性には問題ないことが確認できた。なお、用手法で事前に作製した検量線（図４１ｄ）から、本条件で得られた希釈率は１／５５．３と算出された。

FIG. 41 and Table 3 show the reproducibility evaluation results when the sample was absorbed in the absorbent portion at the tip of the cotton swab (FIG. 9) instead of the liquid sample. A sample prepared by dissolving a freeze-dried HbA1c control (level 2) manufactured by Tosoh in 0.25 mL of pure water was used. 41a is a representative chromatogram, FIG. 41b is a result of plotting the elution time of A1c and A0 for each measurement, FIG. 41c is a result of plotting A1c (%) for each measurement, and FIG. 41d is for calculating the dilution rate. Table 3 summarizes the elution time, peak area, and A1c (%) of A1c and A0 for each measurement. It can be seen that a good chromatogram was obtained even when the sample was loaded and diluted with a cotton swab as a medium, and the diluted specimen was introduced into a liquid chromatograph and analyzed (FIG. 41a). The reproducibility (CV) of elution time of A1c and A0 is 0.71% and 0.44%, respectively, and even if injection is performed through a diluting device, a phenomenon such as sample blanking occurs. Suggests not. The reproducibility (CV) of the peak areas of A1c and A0 was about 13.5% and 13.5%, respectively, which was inferior to the method of directly loading the liquid sample, but the reproducibility of A1c (%) ( CV) was very good at 0.17%, and it was confirmed that there was no problem in the quantitativeness. It should be noted that the dilution rate obtained under these conditions was calculated as 1 / 55.3 from a calibration curve (FIG. 41d) prepared in advance by the usage method.

実施例４
図２７に示す本発明の分析装置のうち、希釈部（１４）を図７ａに示す吸引／吐出の流路が共通の形式（往復動作方式）とし、希釈ポート（３１）を直径５ｍｍΦ×深さ２０ｍｍの円柱形状（図８ａ）（容積は約４００μＬ）とした分析装置（以下、実施例４の分析装置）を用いて、実施例３と同様に、試料の希釈および希釈試料の分析を行なった。

Example 4
In the analyzer of the present invention shown in FIG. 27, the diluting section (14) has the same type (reciprocating operation method) as the suction / discharge flow path shown in FIG. 7a, and the dilution port (31) has a diameter of 5 mmΦ × depth. The sample was diluted and the diluted sample was analyzed in the same manner as in Example 3 using an analyzer (hereinafter, the analyzer of Example 4) having a 20 mm cylindrical shape (FIG. 8 a) (volume is about 400 μL). .

  FIG. 42 and Table 4 show the reproducibility evaluation results when the sample is loaded in a form in which the sample is absorbed in the absorption part at the tip of the test piece (FIG. 11) using the analyzer of the present invention shown in FIG. A sample prepared by dissolving a freeze-dried HbA1c control (level 2) manufactured by Tosoh in 0.25 mL of pure water was used. 42a is a representative chromatogram, FIG. 42b is a result of plotting the elution time of A1c and A0 for each measurement, FIG. 42c is a result of plotting A1c (%) for each measurement, and FIG. 42d is for calculating the dilution rate. Table 4 summarizes the elution time, peak area, and A1c (%) of A1c and A0 for each measurement. It can be seen that a good chromatogram is obtained even when the sample is loaded and diluted with the sample piece as a medium, and the diluted specimen is introduced into the liquid chromatograph and analyzed (FIG. 42a). The reproducibility (CV) of the elution time of A1c and A0 is as good as 0.39% and 0.29%, respectively, and even if injection is performed through a diluting device, a phenomenon such as sample blanking has occurred. Suggests not. The reproducibility (CV) of the peak areas of A1c and A0 was about 16.0% and 15.5%, respectively, which was inferior to the method of directly loading the liquid specimen, but the reproducibility of A1c (%) ( CV) was as good as 1.3%, and it was confirmed that there was no problem in quantitativeness. However, since the reproducibility of A1c (%) is deteriorated as compared with the analyzer shown in FIG. 27 in which the circulation operation method (FIG. 7b) is adopted as the dilution part (14), the absorbent part of the swab or the test piece In the case of measuring by loading a sample that has been absorbed, it is preferable to use an analyzer that employs a circulation operation method (FIG. 7b) in the dilution section (14). Note that the dilution rate obtained under these conditions was calculated to be 1 / 94.0 from the calibration curve (FIG. 42d) prepared in advance by the usage method.

1: Liquid feed pump 2: Sample introduction valve 3: Analysis column 4: Column thermostat 5: Detector 6: Loop 7: Sample (specimen)
8: Eluent (buffer)
9: Washing liquid 10: Syringe 11: Solenoid valve 12: Dilution liquid 13: Flow path switching valve 14: Dilution section 15: Liquid feeding section 16a: Cotton swab 16b: Test piece 17: Dilution liquid feeding section 18: Suction discharge section 19: Suction port 20: Suction / discharge port 21: Check valve 22: Reaction piping 31: Dilution port 32: Drain port 33: Drain port 34: Flow path 41: Diaphragm 42: Piston 43: Spring 44: Capacity adjustment screw 45: Electromagnetic Coil 46: Power supply 51: Manifold 52: Flow path

Claims (4)

A liquid feeding means for feeding the solution;
A loop that can be filled with a sample and a sample filling means that fills the loop with a sample are provided. The sample filling means can fill the loop with the sample and the sample filled in the loop is fed by the liquid feeding means. A sample introduction means capable of switching between a state in which liquid can be fed by the prepared solution;
An analysis device comprising a detection means,
The sample filling means comprises:
A diluent supply for diluting the sample;
A dilution section for loading the sample and diluting the sample with the diluent;
A dilution liquid feeding means having a suction port and a discharge port and capable of feeding the diluted liquid from the suction port to the discharge port, a diaphragm capable of holding the liquid, and a suction discharge port, and the liquid in the diaphragm One or more suction / discharge means capable of suction / discharge, and a liquid-feeding unit capable of sucking and discharging the diluted solution and the diluted sample diluted by the dilution unit,
A device comprising: said device. Liquid feeding means for feeding the first solution;
A loop that can be filled with a second solution and a first filling means that fills the loop with the second solution, a state in which the second solution can be filled into the loop by the first filling means, and the loop A first flow path switching means capable of switching between a state in which the second solution filled in the liquid can be fed by the first solution fed by the liquid feeding means;
A loop capable of filling the nth (n is 3 or more) solution and a (n-1) th filling means for filling the nth solution in the loop are provided, and the (n-1) th filling means The nth solution can be switched between a state in which the n solution can be filled in the loop and a state in which the nth solution filled in the loop can be fed by the first solution fed by the liquid feeding means. 1) the flow path switching means;
A flow path system provided in a loop provided with the (n-1) th flow path switching means in the (n-2) th flow path switching means,
A loop capable of filling a sample and a sample filling means for filling the loop with a sample are provided, and a state in which the sample can be filled by the sample filling means and a sample filled in the loop are fed by the liquid feeding means. A sample introduction means capable of switching between a state in which liquid can be fed by one solution;
Detection means;
The sample filling means comprises:
A diluent supply for diluting the sample;
A dilution section for loading the sample and diluting the sample with the diluent;
A dilution liquid feeding means having a suction port and a discharge port and capable of feeding the diluted liquid from the suction port to the discharge port, a diaphragm capable of holding the liquid, and a suction discharge port, and the liquid in the diaphragm One or more suction / discharge means capable of suction / discharge, and a liquid-feeding unit capable of sucking and discharging the diluted solution and the diluted sample diluted by the dilution unit,
A device comprising: said device. 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 analyzer according to claim 1 or 2, wherein a check valve is provided in each of the first flow path and the second flow path. The analyzer according to any one of claims 1 to 3, wherein the analyzer is a liquid chromatograph.

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