JPH0326349B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a sample injection dilution device, and more particularly to a sample injection dilution device that can be used in a simple biochemical analyzer.

Many biochemical analyzers are currently on the market, but
They often require dilution of the sample. For example, an analyzer that performs blood analysis will always require dilution. In the case of an analyzer that uses a cell in the analysis measurement section, dilution is performed by injecting a precisely quantified trace amount of a sample into a cell containing a buffer solution that has been quantified in advance. In such a case, the amount of sample to be quantified is extremely small, on the order of several tens of microns, so it is necessary to prepare an expensive dedicated quantifier. Furthermore, in the case of an analyzer that uses a pump to quantify a certain amount of sample and then dilutes it by adding a buffer solution, it is necessary to use a very precise pump. If the suction amount and therefore the discharge amount of the pump is on the order of several 100 microns, it is inexpensive and allows accurate quantification. However, pumps capable of accurately quantifying minute amounts of samples as described above are extremely expensive. Therefore, no matter which method is used in the past, when conducting a simple analysis, the means for injecting and diluting the sample become expensive, making the analyzer expensive as a whole.

Therefore, the present invention provides an injection dilution device that can more easily perform accurate injection dilution of a sample.

Simply put, the present invention is such that a first inflow channel communicating with a sample and a second inflow channel communicating with a buffer solution merge, an outflow channel extends from there, and a certain amount of water is supplied to the second inflow channel.
A first pump capable of suctioning only (A) is provided, a second pump capable of suctioning a certain amount (B); (B>A) is provided in the outflow path, and the first inflow increases according to the suction of the second pump. This is a sample injection and dilution device in which a predetermined amount (B-A) of a sample is injected from a channel, and a sample diluted to (B-A)/B is obtained in an outflow channel.

The above objects and other objects and features of the invention will become more apparent from the following detailed description with reference to the drawings.

FIG. 1 is a diagram showing one embodiment of the present invention. The injection diluter 1 has a sample table 2 with a horizontal part on which a sample can be placed. A sample 3 is placed on the sample table 2, and a first inflow path 4 is formed in communication with the sample 3 placed on the sample table 2. A second inflow path 5 joins the first inflow path 4 . The other end of the second inflow path 5 communicates with a buffer solution 6 for dilution. Second inflow path 5
A first pump 7 capable of sucking a predetermined amount (A) of the buffer solution 6 is provided in the middle. An outflow path 8 extends from the confluence of the first inflow path 4 and the second inflow path 5, and a stirring section 9 is formed in the middle of this outflow path 8. A second pump 1 is further provided in the outflow path 8 that has passed through the stirring section 9.
0 is provided, and this second pump 10 has a capacity of a certain amount (B). Here, the suction amount B of the second pump 10 is selected to be larger than the suction amount A of the first pump 7, and the difference (B-A) is selected as the amount of sample to be injected. The other end of the outflow channel 8 is a drain 11
brought to you.

The outflow path 8 is provided with an enzyme electrode 12 as an example of a measuring means. This enzyme electrode 12 has an immobilized enzyme membrane 13 that can come into contact with the solution passing through the outflow channel 8 . This immobilized enzyme membrane 13 has an enzyme immobilized thereon that can specifically react with a specific component or substrate contained in the solution passing through the outflow path 8 . The immobilized enzyme membrane 13 is associated with a polarographic electrode capable of amperometrically detecting substances accompanying the enzyme reaction. The polarographic electrode includes a reference electrode 14 and a working electrode 15. A constant voltage circuit 14 is connected between the reference electrode 14 and the working electrode 15.
A constant polarographic potential is applied by a. The working electrode 15 has a current-voltage conversion circuit 1
5a is connected, and the signal current flowing through this working electrode 15 is converted into voltage. Note that the functions of these enzyme electrodes 12 will be explained in detail later.

In the above configuration, the sample 3 is placed on the sample table 2 of the injection diluter 1. Sample 3
is held in a fixed shape as shown by its surface tension. First and second pumps 7 and 1
0 is driven. Depending on the driving of the second pump 10, the outflow path 8 will have a constant flow rate (B). On the other hand, a predetermined amount (A) of buffer solution 6 flows into the second inflow path 5 by the first pump 7 . Therefore, the difference (B-A) between the suction amount B of the second pump 10 and the suction amount A of the first pump 7 increases on the sample table 2.
The sample 3 placed thereon will flow into the first inflow path 4 . Therefore, in the outflow path 8, (B-
A diluted sample solution is obtained with a dilution ratio expressed as A)/B. The amount of such a sample (B-A) should be such that the solution can flow continuously through that part for the time period necessary for analysis by the enzyme electrode 12, for example, at least several tens of microns is required. Will. However, even if the amount of the sample is larger than that, (B-A)/B remains constant, so the dilution ratio does not change and the analytical measurements are not affected.

Once the sample 3 on the sample table 2 has been injected and disappears from the sample table 2, the next sample is simply placed on the sample table 2, and the above-described injection dilution operation is repeated. If the sample on the sample table 2 disappears, the outflow channel 8 will contain air bubbles together with the buffer solution, but this will not have any effect on the measuring means, such as the enzyme electrode 12. Rather, it was found that such bubbles have a cleaning effect on the enzyme electrode. Therefore, the timing of supplying or placing the sample 3 on the sample table 2 is completely free, and analysis and measurement can therefore be performed at any time.

As mentioned above, in this embodiment the second pump 10
The suction amount (B) by the first pump 7 and the suction amount (A) by the first pump 7 are always kept at a constant ratio (B-A)/B. To do so, for example, a pump as shown in FIG. 2 may be used. The first pump 7 and the second pump 10 are rotationally driven by a common induction motor 16. That is, the output shaft 1 of the common induction motor 16
7, two sets of two discs are fixed so that they can rotate together. Between each pair of discs,
A plurality of squeezing rollers 18 and 19 are provided at equal intervals. Flexible tubes such as silicone tubes 5a and 8a are wound around these squeezing rollers, respectively. Note that the tubes 5a and 8a are assumed to have the same inner diameter here. Therefore, the ratio of the flow rates of the two pumps 7 and 10 is determined by the radii r1 and r2 (second
Figure). Therefore, by appropriately setting such squeezing radii r1 and r2, two pumps with different flow rates and a constant flow rate ratio can be easily obtained. Therefore, this second
In the illustrated example, in addition to the advantage that only one motor is required for driving, even if the rotation speed of the motor 16 changes, the flow rates of the two pumps 7 and 10 change, but the ratio of the flow rates does not change, which has the advantage that the dilution ratio also does not change as a result. For example, if the first pump 7 is
For example, 200μ flows into the second pump 10.
flows, the outflow path 8 has (B-A)/
B, that is, a (200-190)/200=1/20 times diluted solution is obtained. The rotational speed of the motor 16 is increased by 10% to cause the first pump 7 to e.g.
209μ flow, for example 220μ to the second pump 10
Even if it flows, the dilution ratio is (220−209)/220
= 1/20, and it can be seen that the dilution ratio is constant regardless of fluctuations in the rotation speed of the motor 16. In one experiment, silicon tubes with an inner diameter of 0.5 mm and an outer diameter of 1.0 mm were used as tubes 5a and 8a.
The stroke radius r2 was set to 1.8 cm, and the stroke radius r1 of one side was set to 2.0 cm. In addition, each pump 7 and 1
Although it is possible to always obtain a fixed ratio of suction amount by using separate motors such as pulse motors for each motor, according to the embodiment shown in FIG. 2, the same effect can be obtained with an inexpensive induction motor. .

In the experiment, rat blood analysis was performed using the system shown in Figure 1. That is, using rat blood, the blood is diluted approximately 20 times and sent to the enzyme electrode 12 where glucose is determined, and the blood glucose concentration (blood sugar level) contained in the rat blood is determined.
Measurements were made. Sample i.e. rat blood 3
was supplied onto the sample table 2 in an amount of about 20μ so as to block the first outflow path 4 located approximately in the center of the sample table 2, and left there. Rat blood 3 was on the sample table 2 for about 1 minute. During that time, the rat blood 3 was sucked in fixed amounts through the first inflow path 4 and diluted, decreasing at a constant rate. Since the time required for the response of the enzyme electrode 12 to detect glucose was approximately 30 seconds, it is understood that the rat blood sample must be placed at least 10μ.
Note that the dilution buffer supplied through the first pump 7 was approximately 20 c.c. per hour. When the sample tends to remain, such as blood, the length of the first inlet channel 4 is set to 1.5 mm in order to prevent the sample from blocking the first inlet channel 4 and to clean the sample well. However, the results were good. The diluted rat blood is supplied to the enzyme electrode 12 after passing through the stirring section 9 by the suction force of the second pump 10. The diluted and stirred rat blood flows in contact with an immobilized enzyme membrane 13 on which glucose oxidase (GOD) is immobilized, and then passes through a pump 10 and is drained into a drain 11.

Enzyme GOD performs the following enzymatic reaction when the substrate glucose is present in rat blood.

Glucose + O ₂ GOD ――――→ Gluconic acid + H ₂ O ₂ Enzyme GOD has extremely high substrate specificity that does not react at all with substrates other than glucose in rat blood; The amount of enzyme consumed or hydrogen peroxide produced is proportional to the concentration of the substrate glucose. Therefore, by attaching the GOD-immobilized enzyme membrane 13 to the surface of the polarographic electrodes 14 and 15, it is possible to reduce the amount of enzyme that diffuses from the electrode surface or increase the amount of hydrogen peroxide that diffuses to the electrode surface during the enzyme reaction. 14,
By measuring in step 15, the concentration of glucose contained in rat blood can be determined. In the experiment, the polarographic electrodes 14 and 15 measured the increase in hydrogen peroxide. For this purpose, reference electrode 1
4 was a silver electrode, and the working electrode 15 was platinum. A DC polarographic potential of +0.7 V was applied to the platinum working electrode 15 with respect to the silver reference electrode 14 by the constant voltage circuit 14a. In addition, the immobilized enzyme membrane 13
The enzyme GOD was immobilized on a thin film of acetylcellulose or polycarbonate by a cross-linking immobilization method using glutaraldehyde. The polarographic reaction on the surfaces of the reference electrode 14 and the working electrode 15 is expressed by the following equation.

Reference electrode: O ₂ +4H ⁺ +4e ^- →2H ₂ O Working electrode: H ₂ O ₂ →2H ⁺ +O ₂ +2e ^- A voltage proportional to the signal current flowing through the working electrode 15 is detected by the current-voltage conversion circuit 15a. do. Therefore, the voltage from this circuit 15a is obtained as being proportional to the concentration of the substrate, ie, glucose, contained in the solution, ie, rat blood.
Therefore, such glucose concentration can be detected by recording such voltage output on a recorder (not shown). FIG. 3 shows an example of experimental results recorded by a recorder, in which the same rat blood was measured three times, and almost the same output voltage was obtained each time.

In addition, in the above-mentioned Example, the measuring means was arranged in the outflow path 8 upstream of the second pump 10. However, it goes without saying that this may be placed downstream of the second pump 10.

Furthermore, as the measuring means, various component analysis or measuring instruments can be used in addition to the enzyme electrode used in the examples.

As described above, according to the present invention, a sample can be injected and diluted with high precision using a simple configuration. Such an infusion diluter can be obtained at a lower cost since it does not require the use of a pump with a low flow rate and high accuracy.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a system according to an embodiment of the present invention. FIG. 2 is an illustrative view showing an example of a pump that can be used in the embodiment shown in FIG. FIG. 3 is a polarogram showing an example of experimental results using the embodiment shown in FIG. In the figure, 1 is an injection diluter, 2 is a sample table, 3 is a sample, 4 is a first inflow path, 5 is a second inflow path, 6 is a buffer solution, 7 is a first pump, 8 is an outflow path,
10 is a second pump, and 12 is an enzyme electrode.

Claims (1)

[Scope of Claims] 1. A sample placement section having a horizontal portion on which a sample can be placed, a first inflow communicating with the horizontal portion of the sample placement section and introducing the sample placed there. a second inflow path that communicates with the dilution buffer solution and combines the liquid with the first inflow path; an outflow path extending from the confluence of the first and second inflow paths; provided in the middle of the second inflow path; a first pump that sucks a certain amount (A) of the buffer solution, and a first pump that is provided in the middle of the outflow path and sucks out a certain amount (B) that is larger than the certain amount (A) by a predetermined amount. The predetermined amount (B-A) of the sample is injected from the first inflow path in response to the suction of the second pump, and thereby the predetermined amount (B-A) of the sample is injected into the outflow path. ) / B diluted sample is obtained. 2. The first and second pumps are driven by a common motor, and the first and second pumps aspirate buffer solution and diluted sample, respectively, at a fixed ratio. sample injection diluter. 3. The first pump includes a first flexible tube through which a buffer solution passes, and is rotationally driven by the common motor to rotate the first flexible tube to a certain length according to the rotation. The first step to mastering your job
a rubbing roller; the second pump includes a second flexible tube through which the diluted sample passes; and a second flexible tube that is rotationally driven by the common motor and responsive to the rotation of the second flexible tube. 3. The sample injection and dilution device according to claim 2, further comprising a second squeezing roller for squeezing the flexible tube into sections of a predetermined length. 4. The first and second pumps are rotationally driven by different motors, so that the first and second pumps suck the buffer solution and the diluted sample, respectively, at a fixed ratio. A sample injection dilution device according to scope 2. 5. Any one of claims 1 to 4, comprising a measuring means provided in the outflow path for measuring the concentration of a specific component contained in the diluted sample obtained in the outflow path. A sample injection dilution device as described above. 6. The measurement means includes: an immobilized enzyme membrane on which an enzyme that specifically reacts with a specific component contained in the diluted sample is immobilized; and a polarographic electrode that is sensitive to the reaction between the enzyme and the specific component. Claim 5, including:
Sample injection dilution device as described in section.