JP5637300B2 - Sample solution concentration measuring method and sample solution concentration measuring apparatus - Google Patents

Description

  The present invention relates to a sample solution concentration measuring method for quantifying the concentration of a sample solution by measuring chemiluminescence generated by mixing the sample solution and the reactant solution, and a sample solution concentration measuring apparatus used therefor.

  In the artificial dialysis medical treatment, a tubular hollow fiber assembly called a dialyzer made of a semipermeable membrane having an inner diameter of about 100 μm is used. Solutes such as urea present in the blood are removed from the body by permeating into the dialysate side while passing through the dialyzer. Conventionally, the amount of urea in blood has been analyzed by collecting blood, and has been evaluated as a value of blood urea nitrogen (hereinafter sometimes abbreviated as “BUN”).

  As a method for measuring the urea concentration in a sample solution containing urea, a colorimetric method that measures the urea concentration by using a reagent that develops color by reacting with urea and comparing it with the standard color of the reagent, specific to urea An enzyme thermistor method that measures urea concentration by immobilizing urease, which is a working enzyme, around glass beads and measuring the heat of reaction accompanying the hydrolysis reaction of urea, an oxidizing agent that reacts with urea to produce chemiluminescence, for example A chemiluminescence method for measuring urea concentration based on the intensity of chemiluminescence (hereinafter sometimes abbreviated as “CL”) using hypohalite is known.

  In the colorimetric method, there is a problem in that it takes time to prepare a reagent that changes color by reacting with urea, which is one of the causes of measurement errors and that it takes time to complete the measurement. Moreover, it was not suitable for monitoring the change in concentration over time.

  In the enzyme thermistor method, the enzyme deteriorates each time the measurement is repeated, the change with time of the enzyme is large, and it is difficult to measure stably for a long period of time. Although it can be used for monitoring the urea concentration, there is a problem in accuracy when used for multiple measurements.

  Chemiluminescence is said to occur when excited nitrogen generated in the process of urea and hypohalite ion reaction returns to the ground state (Non-patent Document 1). Since the chemiluminescence method can measure the urea concentration in real time, if the sample solution is a sample solution containing urea, it should be used as a means of knowing when to end dialysis treatment in artificial dialysis medicine. Can do. For example, Non-Patent Document 2 describes measuring the urea concentration in the dialysis waste liquid as a guide for knowing the BUN value. Specifically, it describes a method for measuring urea concentration by measuring chemiluminescence generated by the reaction of urea in dialysis waste liquid with sodium hypobromite. However, chemiluminescence efficiency and reproducibility are not always good, and improvements have been demanded.

  In addition, a chemiluminescence measuring method (flow injection method) using a reaction vessel obtained by processing a glass tube into a spiral shape is known. This is because the sample solution and the reactant solution are sent to the inlet of the reaction vessel by a pump or the like, and the sample solution and the reactant solution are mixed and introduced into the spiral tube at the inlet, and the generated chemiluminescence is measured. Is the method. However, there are cases where the sample solution and the reactant solution cannot be mixed uniformly in a short time. Especially when the sample solution and the reactant solution are mixed, a reaction with a short chemiluminescence lifetime occurs or bubbles are generated. When such a reaction occurs, the reproducibility of the measurement of the chemiluminescence intensity is not good and an improvement has been demanded.

  Patent Document 1 discloses a chemiluminescence measurement method for measuring chemiluminescence generated in a cylinder, in which one of a sample solution and a reactant solution is sucked into the cylinder by moving a piston in the cylinder, and then Chemiluminescence characterized by inhaling the other solution and generating a turbulent flow in the cylinder by the jet of the other solution to uniformly mix the sample solution and the reactant solution. The measurement method is described. According to this method, chemiluminescence measurement with high luminous efficiency and good reproducibility can be performed. However, in the above-described method, liquid leakage occurs from the piston sliding part due to repeated use many times, so that it is often necessary to replace the piston, and a large amount of the reagent solution is used, so a large amount of the reagent solution needs to be stocked. In addition, since a precise piston sliding mechanism is required, the cost may be increased, and improvement has been demanded.

  Patent Document 2 includes a plurality of independent reaction chambers formed by anisotropic etching on the surface of a silicon substrate, and a flat plate that is anodically bonded to the surface of the silicon substrate and seals the reaction chamber. The bioreactor microreactor is characterized by the following: According to this, for example, a large number of biochemical reactions of 1000 or more can be performed simultaneously in parallel, and not only simple analysis but also substance synthesis reactions such as protein synthesis can be performed on the cell. ing. In addition, it is said that biochemical reactions can be optically observed and the emission intensity can be monitored. However, in the above microreactor aiming at finally obtaining a reaction product, there are cases where uniform mixing cannot be performed in a short time in each independent reaction chamber. In particular, if a reaction with a short chemiluminescence lifetime occurs or a reaction that generates bubbles occurs when the sample solution and the reagent solution are mixed, the chemiluminescence reaction in each independent reaction chamber occurs. The reproducibility was not always good, and improvement was demanded.

International Publication No. 2009/035008 JP 10-337173 A

Xincheng Hu et al., “Bull. Chem. Soc. Jpn.”, 1996, Vol.69, No.5, p.1179-1185 Toru Okabayashi et al., “Imperial Dialysis”, 2006, Vol. 22, No. 8, p.1199-1204

  The present invention has been made to solve the above-mentioned problems, and it is possible to accurately determine the concentration of a sample solution by measuring the chemiluminescence intensity generated by mixing the sample solution and the reagent solution with good reproducibility. It aims at providing the measuring method which can be performed. Moreover, it aims at providing the suitable use of such a measuring method.

  An object of the present invention is to provide a sample solution concentration measurement method for quantifying the concentration of a sample solution by measuring the chemiluminescence intensity generated in the reaction vessel, wherein the reaction vessel has 2 reaction tanks each having an ejection hole and a discharge hole. Two or more are connected in series, and the cross-sectional area ratio (S1 / S2) between the cross-sectional area (S1) of each of the reaction vessels and the cross-sectional area (S2) of the ejection hole is 3 or more, The solution and the reactant solution are introduced into the first reaction tank from the ejection hole of the first reaction tank, and a turbulent flow is generated in the first reaction tank by the jet of the introduced solution to react the sample solution with the sample solution. The first mixed solution obtained is mixed into the second reaction tank through the discharge hole of the second reaction tank through the discharge hole of the first reaction tank, and the first mixed solution is mixed with the agent solution. The turbulent flow is generated in the second reaction tank by the jet flow of And the reaction vessel optionally has a configuration in which one or more additional reaction vessels are repeatedly arranged downstream of the second reaction vessel, and the above in the second reaction vessel. The mixed solution may be further mixed in the same manner as the mixing, and this is solved by providing a sample solution concentration measuring method characterized by measuring the chemiluminescence intensity generated in the reaction vessel.

  At this time, it is preferable that the sample solution is a sample solution containing urea, and it is preferable that the sample solution is a dialysis waste liquid. It is also preferable that the reactant solution is a reactant solution containing hypohalite ions.

  Furthermore, the above-described problem is a sample solution concentration measuring apparatus for quantifying the concentration of a sample solution by measuring the intensity of chemiluminescence generated in a reaction vessel, wherein two or more reaction tanks having ejection holes and discharge holes are connected in series. A reaction vessel connected to each other, the cross-sectional area ratio (S1 / S2) between the cross-sectional area (S1) of each of the reaction vessels and the cross-sectional area (S2) of the ejection holes is 3 or more, and the reaction vessel A means for introducing a sample solution and a reagent solution into the first reaction tank from the ejection holes of the first reaction tank, wherein at least a part of the transparent part has a transparent part, and a photodetector is disposed outside the transparent part. A turbulent flow is generated in each of the reaction tanks by a jet of the introduced solution, and the chemiluminescence intensity generated in the reaction vessel is measured by mixing the sample solution and the reactant solution. A sample solution concentration measuring device characterized by Also solved by subjecting.

  At this time, it is preferable to have at least one reaction tank in which an angle α between the axis of the ejection hole and the axis of the discharge hole is 15 degrees or more and 165 degrees or less, and quantifying the urea concentration is the present invention. This is a preferred embodiment. An artificial dialysis apparatus that quantifies the urea concentration in the dialysis waste liquid by the sample solution concentration measuring apparatus is a preferred embodiment of the present invention.

  In the sample solution concentration measuring method of the present invention, the mixing of the sample solution and the reactant solution is repeated by the jet of the solution introduced into each reaction tank. Thereby, since the chemiluminescence intensity generated by the mixing can be measured with high reproducibility, the concentration of the sample solution can be accurately quantified. Even when the reaction rate of chemiluminescence is fast, the chemiluminescence intensity can be measured with good reproducibility. In particular, the chemiluminescence intensity can be measured with good reproducibility even when bubbles are generated along with the chemiluminescence reaction when the sample solution and the reactant solution are mixed. Further, when the sample solution is a sample solution containing urea, the urea concentration can be measured in real time, so that it can be suitably used as an artificial dialysis apparatus that can know the end of the dialysis time.

It is the schematic plan view which showed an example of the sample solution concentration measuring apparatus of this invention. It is the schematic side view which showed an example of the sample solution concentration measuring apparatus of this invention. 2 is a chemiluminescence response waveform diagram obtained in Example 1. FIG. FIG. 3 is a chemiluminescence response waveform diagram obtained when the concentration of an aqueous urea solution was changed in Example 1. FIG. 2 is a chemiluminescence response waveform diagram used for calculation of chemiluminescence lifetime τ in Example 1. FIG. 3 is a correlation diagram between the urea concentration obtained in Example 1 and the chemiluminescence intensity. 6 is a chemiluminescence response waveform diagram obtained in Example 2. FIG. 6 is a chemiluminescence response waveform diagram obtained in Comparative Example 1. FIG. It is a schematic plan view of the apparatus which measures the chemiluminescence intensity which arose in each reaction tank AG. It is a chemiluminescence response waveform diagram obtained by measuring the chemiluminescence intensity generated in each reaction vessel A-G.

  Hereinafter, the present invention will be described more specifically with reference to the drawings. FIG. 1 is a schematic plan view showing an example of a sample solution concentration measuring apparatus 1 used in the present invention, and FIG. 2 is a schematic side view showing an example of the apparatus 1. In FIG. 1, a reaction vessel 2 is provided in which reaction vessels having ejection holes and discharge holes from the first reaction vessel A to the seventh reaction vessel G are connected in series. Each reaction vessel has a transparent portion on the upper surface side of the reaction vessel 2, and a photodetector 24 is arranged at a position facing the upper surface of the reaction vessel 2 as shown in FIG. 2. The sample solution inlet 3 and the first reaction tank A are connected by a pipe 7. The sample solution is introduced into the first reaction tank A from the ejection hole 9 of the first reaction tank A through the pipe 7 from the sample solution introduction port 3 by operating the pump 5. On the other hand, the reactant solution inlet 4 and the first reaction tank A are connected by a pipe 8. The reactant solution is introduced into the first reaction tank A from the ejection hole 9 of the first reaction tank A through the pipe 8 from the reactant solution inlet 4 by operating the pump 6. Then, a turbulent flow is generated in the first reaction tank A by the jet of the solution introduced into the first reaction tank A, and the sample solution and the reactant solution are uniformly mixed, so that the first mixed solution becomes can get. The obtained first mixed solution is introduced into the second reaction tank B from the ejection hole 11 of the second reaction tank B through the discharge hole 10 of the first reaction tank A. A turbulent flow is generated in the second reaction tank B by the jet flow of the introduced first mixed solution, and the first mixed solution is further mixed to obtain a second mixed solution. Next, the obtained second mixed solution is contained in each reaction tank from the third reaction tank C to the seventh reaction tank G provided downstream of the discharge hole 12 of the second reaction tank B. Further mixed. In this way, the chemiluminescence intensity generated by mixing the sample solution and the reagent solution in the reaction vessel 2 is measured by the photodetector 24, and the concentration of the sample solution can be quantified.

  The sample solution concentration measuring apparatus 1 of the present invention includes a reaction vessel 2 in which two or more reaction vessels having ejection holes and discharge holes are connected in series. By setting it as such a structure, the reproducibility of the measurement of the chemiluminescence intensity generated in the reaction vessel 2 becomes good. As can be seen from the results of Comparative Example 1 described later, when a reaction vessel having only one reaction vessel was used, the stability of the chemiluminescence intensity generated by mixing the sample solution and the reactant solution was not good. . Therefore, it is significant to employ the reaction vessel 2 in which two or more reaction vessels having ejection holes and discharge holes are connected in series. In the present invention, the reaction vessel 2 may optionally have a configuration in which one or more additional reaction vessels are repeatedly arranged downstream of the second reaction vessel B. Thus, the reproducibility of the measurement of the chemiluminescence intensity is improved by repeatedly arranging the plurality of reaction vessels. From this point of view, as the reaction vessel 2 used in the present invention, it is preferable that three or more of the reaction vessels are connected in series, more preferably four or more are connected in series, and more than five are connected in series. More preferably, it is connected to. In particular, when the concentration of the sample solution is low, the reproducibility of the measurement of chemiluminescence intensity is better when the number of connected reaction vessels is larger.

The shape of the reaction vessel used in the present invention is not particularly limited. The shape of the reaction vessel may be a cylindrical shape, a spherical shape, or a square shape. From the viewpoint of good stirring efficiency, a cylindrical or spherical reaction vessel is preferably used. The size of the reaction vessel used in the present invention is not particularly limited, but from the viewpoint of efficiently generating turbulent flow by the jet of the solution and reducing the amount of the reactant solution used, the capacity of one reaction vessel Is preferably 300 mm ³ or less, more preferably 200 mm ³ or less, and even more preferably 100 mm ³ or less.

  The sample solution concentration measuring apparatus 1 of the present invention preferably has at least one reaction tank in which an angle α between the axis of the ejection hole and the axis of the discharge hole is 15 degrees or more and 165 degrees or less. By adopting such a configuration, the stability of measurement of the chemiluminescence intensity generated in the reaction vessel 2 becomes good. For this reason, the inventors collide with the inner wall of the downstream reaction tank when the jet of the solution is introduced from the discharge hole of the upstream reaction tank to the ejection hole of the downstream reaction tank. This is presumed to result in more uniform mixing.

  In the sample solution concentration measuring apparatus 1 of the present invention, the cross-sectional area ratio (S1 / S2) between the cross-sectional area (S1) of each reaction vessel and the cross-sectional area (S2) of the ejection hole is 3 or more. Here, the cross-sectional area (S1) of the reaction tank means the maximum cross-sectional area in a plane perpendicular to the water flow flowing into the reaction tank from the ejection holes. When the cross-sectional area ratio (S1 / S2) is 3 or more, when the sample solution and the reactant solution are introduced into the reaction tank from the ejection holes, a jet flow is generated by the solution, and a turbulent flow is generated by the jet flow. It will be. As a result, the sample solution and the reactant solution are rapidly mixed and homogenized, so that fluctuations in the chemiluminescence intensity when measuring the generated chemiluminescence intensity are reduced, the S / N ratio is improved, and the chemiluminescence is increased. The reproducibility of strength measurement is good. When the cross-sectional area ratio (S1 / S2) is less than 3, turbulent flow and laminar flow are mixed when the sample solution and the reactant solution are introduced into the reaction vessel from the ejection holes, and the chemiluminescence intensity is measured. The reproducibility may be reduced, and the cross-sectional area ratio (S1 / S2) is preferably 4 or more, and more preferably 5 or more. On the other hand, the cross-sectional area ratio (S1 / S2) is usually 1000 or less.

  In the sample solution concentration measuring apparatus 1 of the present invention, at least a part of each reaction tank has a transparent portion, and a photodetector 24 is disposed outside the transparent portion. The light detector 24 measures the chemiluminescence generated in the reaction vessel 2 by mixing the sample solution and the reactant solution. Thus, the concentration of the sample solution can be quantified by measuring the chemiluminescence intensity using the photodetector 24. The arrangement of the light detector 24 is not particularly limited as long as it is outside the transparent portion of each reaction tank. However, as shown in FIG. 2, the light detector 24 is arranged so as to be in contact with the upper surface of the reaction vessel. It is preferable to do.

  It does not specifically limit as a material which comprises the reaction tank used by this invention. As mentioned above, from the viewpoint that at least a part of each reaction vessel has a transparent part, the material constituting the reaction vessel may be glass, acrylic resin, polystyrene resin, polyvinyl chloride resin, polycarbonate resin, polyester. It may be a transparent resin such as a resin, or a combination of these. When the flow rates of the sample solution and the reactant solution introduced into the reaction vessel are low, the inventors have reproducibility of the chemiluminescence intensity obtained when at least a part of the reaction vessel is made of glass. It was confirmed that was good. On the other hand, a thermoplastic resin is preferably used from the viewpoint of easy molding and low cost. In addition, by using the sample solution concentration measuring apparatus 1 of the present invention for a long period of time, even if contaminants accumulate inside the reaction tank, it can be manufactured at low cost and can be used disposable. . In addition, when the material constituting the reaction vessel is a thermoplastic resin such as an acrylic resin, the present inventors once distribute the strongly alkaline reactant solution into the reaction vessel, and then the sample solution and the reactant. When the chemiluminescence intensity generated by mixing with the solution was measured, it was confirmed that the reproducibility of the obtained chemiluminescence intensity was improved. The reason for this is not necessarily clear, but the present inventors speculate that there is a possibility that the surface in the reaction vessel may be hydrophilized by the reaction solution having strong alkalinity.

  The sample solution concentration measuring apparatus 1 of the present invention includes means for introducing the sample solution and the reactant solution into the first reaction tank A from the ejection holes 9. The introduction means is not particularly limited as long as the sample solution and the reactant solution can be introduced into the first reaction tank A at a constant flow rate. For example, a diaphragm pump, a piston pump, a plunger pump, a gear pump, etc. are mentioned. Among these, a diaphragm pump is preferably used from the viewpoint of good responsiveness and quantitativeness. Since the sample solution concentration measuring apparatus 1 of the present invention does not require any mechanical operation means other than the introduction means, the apparatus configuration is not complicated and the concentration of the sample solution can be determined without maintenance. . In addition, the sample solution concentration measuring apparatus 1 of the present invention can be manufactured at a low cost and hardly break down.

  The sample solution concentration measuring apparatus 1 of the present invention may include a sample solution supply unit capable of supplying a sample solution, for example, a tank in which the sample solution is stored. From the viewpoint of quantifying the concentration of the sample solution in real time, it is preferable that the sample solution supply unit includes means capable of supplying a new sample solution as needed. As will be described later, when the sample solution is a sample solution containing urea, particularly a dialysis waste liquid, a new dialysis waste liquid can be supplied at any time, so that the urea concentration in the dialysis waste liquid can be quantified in real time. This makes it possible to know the timing at which dialysis treatment should be terminated. Moreover, the sample solution concentration measuring apparatus 1 of the present invention may include a reactant solution supply unit capable of supplying a reactant solution, and includes, for example, a tank in which the reactant solution is accommodated.

  In the sample solution concentration measuring method of the present invention, the concentration of the sample solution is quantified by measuring the intensity of chemiluminescence generated in a reaction vessel 2 in which two or more reaction vessels having ejection holes and discharge holes are connected in series. It is characterized by doing. As in the present invention, the turbulent flow is generated in each reaction vessel by the jet of the solution introduced into the reaction vessel, so that the sample solution and the reactant solution are rapidly mixed and uniformed. The fluctuation of the chemiluminescence intensity when measuring the chemiluminescence intensity is reduced, the S / N ratio is improved, and the reproducibility of the chemiluminescence intensity measurement is improved. Thereby, the sample solution concentration can be quantified.

  In the sample solution concentration measuring method of the present invention, as shown in FIG. 1, the sample solution and the reactant solution are introduced into the first reaction tank A, and the first reaction tank is jetted by the introduced solution. A turbulent flow is generated in A, and the sample solution and the reactant solution are uniformly mixed to obtain a first mixed solution. The obtained first mixed solution is introduced into the second reaction tank B from the ejection hole 11 through the discharge hole 10. A turbulent flow is generated in the second reaction tank B by the jet flow of the introduced first mixed solution, and the first mixed solution is further mixed to obtain a second mixed solution. The obtained second mixed solution is further mixed in each reaction tank from the third reaction tank C to the seventh reaction tank G provided downstream of the discharge hole 12. Then, the mixed solution is discharged from the discharge port 23. In this way, the concentration of the sample solution can be quantified by measuring the chemiluminescence intensity generated by mixing the sample solution and the reactant solution in the reaction vessel 2 with the photodetector 24.

  In the present invention, as described above, the mixing in each reaction tank is repeated, whereby the chemiluminescence intensity generated by the mixing can be measured with good reproducibility. In particular, the reproducibility of the measurement of the chemiluminescence intensity is good even when bubbles are generated along with the reaction that generates chemiluminescence by mixing the sample solution and the reactant solution. The present inventors have confirmed that when bubbles are generated when the sample solution and the reactant solution are mixed, the stability of chemiluminescence intensity was not good, and the reaction was inhibited by the generated bubbles. It is presumed that the chemiluminescence is scattered. In the examples described later, the present inventors have shown that bubbles are generated when the sample solution and the reagent solution are mixed in the first reaction tank A to obtain the first mixed solution. It is confirmed visually. When the bubbles generated together with the first mixed solution pass through the respective reaction tanks from the second reaction tank B to the seventh reaction tank G, it is difficult to visually confirm the generated bubbles. It was. This is considered to be because the bubbles generated with the first mixed solution were refined by repeating the mixing by the jet of the solution in each reaction tank. On the other hand, as can be seen from the results of Comparative Example 1 described later, when a reaction vessel having only one reaction vessel is used, the stability of the chemiluminescence intensity generated by mixing the sample solution and the reactant solution is not good. There wasn't. Therefore, it is a preferred embodiment of the present invention to quantify the concentration of the sample solution by measuring the chemiluminescence intensity generated in the reaction vessel 2 when the reaction that generates bubbles occurs.

  The sample solution concentration measurement method of the present invention has good reproducibility of chemiluminescence intensity measurement even when the reaction rate of chemiluminescence generated by mixing the sample solution and the reagent solution is high. Therefore, the concentration of such a sample solution can be quantified with high accuracy. Here, a fast reaction rate of chemiluminescence indicates that the chemiluminescence lifetime τ is small. In the present invention, the chemiluminescence lifetime τ is the chemiluminescence when the decay of the chemiluminescence intensity occurs. It is defined as the time until the intensity decreases to a value of 1 / e. As can be seen from the chemiluminescence response waveform diagram of FIG. 5, the chemiluminescence intensity generated by mixing the sample solution and the reactant solution attenuates exponentially when the supply of the sample solution and the reactant solution is stopped. At this time, the chemiluminescence lifetime τ is obtained until the chemiluminescence intensity on the decay curve decreases to a value of 1 / e in the chemiluminescence response waveform diagram in which the vertical axis in the semilogarithmic graph is chemiluminescence intensity and the horizontal axis is time. It is calculated by the time. Thus, even when the reaction rate of chemiluminescence is fast, the reason why the reproducibility of the measurement of chemiluminescence intensity is good is that turbulent flow is generated in each reaction tank by a jet of solution introduced into the reaction tank. By doing so, the sample solution and the reactant solution are rapidly mixed and homogenized, so that fluctuations in chemiluminescence intensity when measuring the generated chemiluminescence intensity are reduced and the S / N ratio is improved. Conceivable. Therefore, a preferred embodiment of the present invention is a sample solution concentration measurement method in which the concentration of a sample solution is quantified by measuring the intensity of chemiluminescence generated in the reaction vessel 2 in a chemiluminescence reaction having a chemiluminescence lifetime τ of 3 seconds or less. It is an aspect.

The sample solution used in the present invention is not particularly limited as long as it is chemiluminescent by mixing with a reactant solution, but it may be a sample solution containing a biologically derived nitrogen-containing compound such as urea, amino acid, protein, etc. A sample solution containing urea is more preferable. When the sample solution is a sample solution containing urea, the sample solution concentration measuring device 1 of the present invention can be suitably used for various applications as a urea concentration measuring device. In addition, the reagent solution used in the present invention is not particularly limited as long as it is chemiluminescent by mixing with the sample solution, but it reacts with the sample solution containing a biologically derived nitrogen-containing compound such as urea, amino acid, protein and the like. From the viewpoint of chemiluminescence, the reactant solution is preferably a reactant solution containing hypohalite ions. The hypohalite ion is not particularly limited, and examples thereof include hypohalite ions such as FO ⁻ , ClO ⁻ , BrO ⁻ and IO ^{−, and} are selected from hypobromite ions or hypochlorite ions. It is preferable that there is at least one. The reactant solution containing hypohalite ions may be supplied to the apparatus in advance as an aqueous solution containing hypohalite ions, or the aqueous solution containing halogen ions is electrolyzed in the apparatus. You may supply by.

  When the sample solution used in the present invention is a dialysis waste liquid, it can be suitably used as an artificial dialysis apparatus characterized by measuring the urea concentration in the dialysis waste liquid. In particular, since the urea concentration can be measured in real time, it can be suitably used as an artificial dialysis apparatus that can know the timing at which dialysis treatment should be terminated. Moreover, since it can also be used for quality and performance evaluation of a dialyzer, the method for evaluating the performance of a dialyzer using the sample solution concentration measuring apparatus 1 of the present invention is also a preferred embodiment of the present invention.

  Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1
Using the sample solution concentration measuring apparatus 1 (vertical: 74 mm, horizontal: 100 mm, height: 74 mm) shown in FIGS. 1 and 2, the concentration of the sample solution was measured. FIG. 1 is a schematic plan view showing an example of a sample solution concentration measuring apparatus 1, and FIG. 2 is a schematic side view of the apparatus 1. As shown in FIG. 1, the reaction vessel 2 (vertical: 40 mm, horizontal: 40 mm, height: 5 mm) has a cylindrical reaction vessel (diameter) from the first reaction vessel A to the seventh reaction vessel G. : 3 mm, height: 3 mm) are connected in series. Glass plates were placed on the upper and lower surfaces of the acrylic resin plate on which the reaction vessels from the first reaction vessel A to the seventh reaction vessel G were formed. Thereby, the upper surface and the lower surface of each reaction tank were made of glass, and the side surfaces of each reaction tank were made of acrylic resin. Each reaction tank from the first reaction tank A to the seventh reaction tank G is provided with an ejection hole (inner diameter: 1 mm) and a discharge hole (inner diameter: 1 mm). The cross-sectional area ratio (S1 / S2) between the cross-sectional area (S1) of each reaction tank and the cross-sectional area (S2) of the ejection hole was 9. The discharge hole 10 of the first reaction tank A and the ejection hole 11 of the second reaction tank B are connected via a narrow passage, and a third reaction is further provided downstream of the discharge hole 12 of the second reaction tank B. Respective reaction tanks from the tank C to the seventh reaction tank G are connected in series. At this time, the angle α in each reaction tank is 90 degrees in the reaction tank A, 120 degrees in the reaction tank B, 60 degrees in the reaction tank C, 180 degrees in the reaction tank D, 60 degrees in the reaction tank E, and 60 degrees in the reaction tank F. The reaction tank G was 120 degrees and 120 degrees. As shown in FIG. 2, a photosensor module, which is a photodetector 24, was attached at a position facing the upper surface of the reaction vessel 2 so as to be in contact with the upper surface.

  The sample solution inlet 3 is connected to the first reaction tank A via a pipe 7 (inner diameter: 1 mm), and the reactant solution inlet 4 is connected to the first reaction tank A via a pipe 8 (inner diameter: 1 mm). It is connected. The sample solution is introduced into the first reaction tank A from the sample solution introduction port 3 through the pipe 7 by operating the pump 5. On the other hand, the reactant solution is introduced into the first reaction tank A from the reactant solution inlet 4 through the pipe 8 by operating the pump 6. As shown in FIG. 1, the configuration in which the pipe 7 and the pipe 8 merge immediately before the sample solution and the reactant solution are introduced into the first reaction tank A from the ejection holes 9 of the first reaction tank A. have. A 9 mM urea aqueous solution was used as a sample solution, and a mixed solution containing 0.5 M hypobromite and 0.2 M sodium hydroxide was used as a reactant solution. When the pump 5 and the pump 6 are operated, the sample solution and the reactant solution are introduced into the first reaction tank A from the ejection holes 9 of the first reaction tank A. A turbulent flow was generated in the first reaction tank A by the jet of the introduced solution, and the sample solution and the reactant solution were uniformly mixed to obtain a first mixed solution and nitrogen gas was generated.

  The obtained first mixed solution is introduced into the second reaction tank B from the ejection hole 11 through the discharge hole 10. A turbulent flow is generated in the second reaction tank B by the jet flow of the introduced first mixed solution, and the first mixed solution is further mixed to obtain a second mixed solution. At this time, the nitrogen gas introduced together with the first mixed solution from the ejection hole 11 of the second reaction tank B becomes fine bubbles by the jet flow of the first mixed solution and is dispersed in the second mixed solution. It was done. The obtained second mixed solution is further mixed in each reaction tank from the third reaction tank C to the seventh reaction tank G provided downstream of the discharge hole 12 of the second reaction tank B. Is done. At this time, nitrogen gas became finer bubbles with mixing in each reaction tank. In this way, the chemiluminescence intensity generated by mixing the sample solution and the reactant solution in the reaction vessel 2 was measured by the photodetector 24, and the concentration of the sample solution was quantified. FIG. 3 shows a chemiluminescence response waveform diagram obtained when both the sample solution and the reactant solution are introduced into the reaction vessel 2 while changing the sample solution and the reactant solution at the same flow rate. FIG. 4 shows a chemiluminescence response waveform diagram obtained when the concentration of the urea aqueous solution is changed. A chemiluminescence response waveform diagram used for calculation of the chemiluminescence lifetime τ is shown in FIG. At this time, the chemiluminescence lifetime τ was 0.9 seconds. FIG. 6 shows a correlation diagram between the urinary concentration and the chemiluminescence intensity.

Here, when the total flow rate Q of the sample and the reagent in the reactor is 40 ml / min (0.667 × 10 ⁻⁶ m ³ / s), the inner radius of the ejection hole is cut off from 0.5 × 10 ⁻³ m. Since the area S is (0.5 × 10 ⁻³ ) ² × 3.14 = 7.85 × 10 ⁻⁷ m ² , the average flow velocity v in this part is v = Q / S = 0.667 × 10. ⁻⁶ / 7.85 × 10 ⁻⁷ = 0.85 m / s. Since the kinematic viscosity coefficient when the sample solution is 20 ° C. water is 1.0 × 10 ⁻⁶ m ² / s, the Reynolds number Re = Ud / ν = 0.85 × 1 × 10 ⁻³ / 1. 0 × 10 ⁻⁶ = 850 <2320 (critical Reynolds number of flowing water in the pipe), and no turbulent flow occurs in the pipe having an inner diameter of 1 mm. However, it is generally known that when a cylinder is placed perpendicular to the flow in the water flow, the flow separates from the cylinder and a vortex is generated from about Re = 23. Therefore, even when the solution is introduced into the cylindrical reaction tank having a diameter of 3 mm and a height of 3 mm from the injection hole having an inner diameter of 1 mm as in this example, the flow due to the solution is separated from the injection hole to generate a vortex, It is inferred that turbulence was generated by the vortex. As a result, it is considered that the sample solution and the reactant solution were uniformly mixed. In addition, the volume of the reaction tank used in this example is 1.5 ² × 3 × 3.14 = 21.2 mm ³ , and seven reactors from the first reaction tank A to the seventh reaction tank G are used. Since the reaction vessels are connected in series, the total volume of the reaction vessel 2 is about 150 mm ³ (1.5 × 10 ⁻⁷ m ³ ). Therefore, the time in which the solution having a flow rate of 0.667 × 10 ⁻⁶ m ³ / s stays in the reaction vessel 2 is about 0.22 seconds, which is necessary for the chemiluminescence reaction between urea and hypobromite ions. Since the time is about several hundreds of milliseconds, it is considered that almost all urea is consumed in the reaction vessel 2 and chemiluminescence occurs.

Example 2
In Example 1, the reaction was carried out in the same manner as in Example 1 except that all the reaction tanks including the upper and lower surfaces of each reaction tank from the first reaction tank A to the seventh reaction tank G were made of acrylic resin. The chemiluminescence intensity generated by mixing the sample solution and the reactant solution in the container 2 was measured by the photodetector 24 to quantify the concentration of the sample solution. FIG. 7 shows a chemiluminescence response waveform diagram obtained when both the sample solution and the reactant solution are introduced into the reaction vessel 2 at the same flow rate. In FIG. 7, when the flow rate is as small as 10 ml / min, the fluctuation of the optical sensor output is more severe than the case of higher flow rates (20 ml / min, 30 ml / min, 40 ml / min), and the measured value of chemiluminescence Was relatively unstable. At this time, when the inside of the reaction tank was visually confirmed, bubbles remaining in each reaction tank were observed. It is considered that the bubbles generated by mixing the sample solution and the reactant solution were not sufficiently refined. Therefore, in order to prevent the chemiluminescence reaction from being inhibited by the generated bubbles, or to prevent the chemiluminescence from being scattered, the flow rate at which the generated bubbles are refined, that is, the flow rate, is reduced. It is desirable to be.

Comparative Example 1
In Example 1, instead of using the reaction vessel 2 in which the respective reaction vessels from the first reaction vessel A to the seventh reaction vessel G were connected in series, a reaction vessel (diameter: 10 mm, height: 3 mm, A reaction vessel having only one (made of polyvinyl chloride) was used. A sample was prepared in the reaction vessel in the same manner as in Example 1 except that a mixed solution of 0.5 M sodium hypochlorite, 2 M sodium bromine, and 0.2 M sodium hydroxide was used as the reactant solution. The chemiluminescence intensity generated by mixing the solution and the reactant solution was measured by the photodetector 24, and the concentration of the sample solution was quantified. FIG. 8 shows a chemiluminescence response waveform diagram obtained when both the sample solution and the reactant solution were introduced into the reaction vessel at a flow rate of 20 ml / min.

Example 3
In Example 1, instead of using the reaction vessel 2, each cylindrical reaction vessel from the first reaction vessel A to the seventh reaction vessel G (diameter: 3 mm, height 3 mm, made of polyvinyl chloride (black) ) Was used in a straight line connected through a narrow passage (orifice, diameter 1 mm, length 3 mm). A transparent plate 26 (made of glass) having a thickness of 1 mm was disposed on the upper and lower surfaces of each of the reaction vessels A to G. As shown in FIG. 9, a photodetector 24 (photomultiplier tube) provided with a pinhole 27 equal to the diameter of each reaction vessel at a position facing the upper surface of the reaction vessel 25 is in contact with the upper surface. Arranged. As in Example 1, a dialysate containing 9 mM urea was used as the sample solution, and a mixed solution containing 0.5 M hypobromite and 0.2 M sodium hydroxide was used as the reactant solution. The photodetector 24 was moved from the first reaction tank A to the seventh reaction tank G by the linear slider 28, and the chemiluminescence intensity in each of the reaction tanks A to G was measured. FIG. 10 shows a chemiluminescence response waveform diagram obtained when the flow rates of the sample solution and the reactant solution are 20 ml / min (total flow rate 40 ml / min). As can be seen from the chemiluminescence response waveform diagram of FIG. 10, the chemiluminescence intensity showed the maximum value in the second reaction tank B, and then decreased as the reaction tank became downstream. From this result, it can be seen that it is effective to connect two or more reaction vessels in series via a narrow passage (orifice) in order to obtain a strong chemiluminescence intensity.

DESCRIPTION OF SYMBOLS 1 Sample solution density | concentration measuring apparatus 2 Reaction container 3 Sample solution inlet 4 Reactant solution inlet 5, 6 Pump 7, 8 Pipe A 1st reaction tank B 2nd reaction tank C 3rd reaction tank D 4th Reaction tank E 5th reaction tank F 6th reaction tank G 7th reaction tank 9 Ejection hole of 1st reaction tank 10 Ejection hole of 1st reaction tank 11 Ejection hole of 2nd reaction tank 12 2nd 13 reaction tank discharge hole 14 third reaction tank discharge hole 15 fourth reaction tank discharge hole 16 fourth reaction tank discharge hole 17 fifth reaction tank discharge Hole 18 Fifth reaction tank discharge hole 19 Sixth reaction tank discharge hole 20 Sixth reaction tank discharge hole 21 Seventh reaction tank discharge hole 22 Seventh reaction tank discharge hole 23 Discharge port 24 Photodetector 25 Reaction vessel 26 Transparent plate 27 Pinhole 28 Linear slider

Claims (4)

A urea concentration measurement method for quantifying the urea concentration of a sample solution containing urea by measuring the chemiluminescence intensity generated in a reaction vessel,
The reaction vessel is composed of three or more reaction tanks having ejection holes and discharge holes connected in series via a linear passage, and an angle α between the axis of the ejection hole and the axis of the discharge hole Having at least one reaction vessel having a temperature of 15 degrees or more and 165 degrees or less,
The cross-sectional area ratio (S1 / S2) between the cross-sectional area (S1) of each reaction vessel and the cross-sectional area (S2) of the ejection hole is 3 or more,
A sample solution containing urea and a reagent solution containing hypohalite ions are introduced into the first reaction tank through the ejection holes of the first reaction tank, and are turbulent in the first reaction tank by the jet of the introduced solution. Generating a flow to mix the sample solution and the reactant solution;
The obtained first mixed solution was introduced into the second reaction tank from the ejection hole of the second reaction tank through the discharge hole of the first reaction tank,
A turbulent flow is generated in the second reaction tank by the jet of the first mixed solution, and the first mixed solution is further mixed;
The obtained second mixed solution is further mixed in each reaction tank provided downstream of the discharge hole of the second reaction tank,
A method for measuring urea concentration, comprising measuring chemiluminescence intensity generated in the reaction vessel.   The urea concentration measuring method according to claim 1, wherein the sample solution containing urea is a dialysis waste liquid. A urea concentration measurement device that quantifies the urea concentration of a sample solution containing urea by measuring the chemiluminescence intensity generated in a reaction vessel,
Three or more reaction tanks having ejection holes and discharge holes are connected in series via a linear passage, and an angle α between the axis of the ejection hole and the axis of the discharge hole is 15 degrees or more and 165 degrees or less. A reaction vessel having at least one reaction vessel,
The cross-sectional area ratio (S1 / S2) between the cross-sectional area (S1) of each reaction tank and the cross-sectional area (S2) of the ejection hole is 3 or more, and at least a part of the reaction tank has a transparent portion. And a photodetector is disposed outside the transparent portion,
Means for introducing a sample solution containing urea and a reactant solution containing hypohalite ions into the first reaction tank from the ejection holes of the first reaction tank, and each of the reaction tanks by a jet of the introduced solution An apparatus for measuring urea concentration, characterized in that a chemiluminescence intensity generated in the reaction vessel is measured by generating a turbulent flow and mixing the sample solution and the reactant solution.   An artificial dialysis apparatus for quantitatively determining a urea concentration in a dialysis waste liquid by the urea concentration measuring apparatus according to claim 5.

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