JPS629306B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improvement in a creatinine analyzer for analyzing creatinine contained in biological fluids such as serum.

Analysis of creatinine in biological fluids has become one of the important test items for diagnosing renal disorders, etc., but it is also a testing method often used for emergency testing during surgery. In the conventional creatinine analysis method, an aqueous solution of creatininase, which is a creatinine degrading enzyme, is mixed with a sample, the reaction of formula (1) is carried out, and the generated ammonia gas is measured.

That is, creatininase specifically acts on creatinine to generate N-methylhydantoin and ammonia gas, and this ammonia gas is detected using an ammonia gas electrode.

In such a conventional creatinine analysis method, a predetermined amount of creatininase is added as an aqueous solution to a sample solution to cause it to act, so it is discarded every time a sample is analyzed and cannot be recovered. Therefore, the analysis method consumes a large amount of enzyme and is expensive. In addition, since the pH value of the buffer solution that is optimal for advancing equation (1) is different from the pH value of the buffer solution that is optimal for generating ammonia gas, the buffer solution is switched in the middle of the flow path. However, the analytical operations become complicated, the equipment becomes complicated, and two types of buffer solutions must be prepared.

The purpose of the present invention is to provide a creatinine analyzer that can perform continuous analysis and has low enzyme consumption. , each ammonium ion selective electrode and a comparative electrode are provided together to form a first electrode pair and a second electrode pair, and the signal value from the first electrode pair is transferred to the second electrode pair. The structure is such that the signal holding means holds the sample specimen until it passes through, and detects the difference in concentration of ammonium ions in the buffer solution.

Recently, technology has been developed to immobilize enzymes on membranes, tubes, porous glass beads, etc., but it is possible to repeatedly use the enzyme by immobilizing creatininase on porous glass beads and filling it into an enzyme reactor. It became possible. According to this, the contact reaction between the enzyme and the substrate can be carried out continuously within the enzyme reactor. In addition, if a buffer solution is prepared and placed at the optimal pH value to proceed with the reaction of equation (1) above, ammonium ions that are substantially proportional to the concentration of creatinine will be generated in the reaction equation of equation (2). . Therefore, by using an ammonium ion selective electrode, ammonium ions can be accurately measured and the concentration of creatinine can also be determined. Note that it is sufficient to use one type of buffer solution with a pH value suitable for proceeding with the above equation (1), so there is no need to switch the buffer solution and distribute it.

FIG. 1 is a system diagram of a creatinine analyzer which is an embodiment of the present invention. 1 is a buffer solution tank, 2 is a liquid pump, 3 is a sample introduction part, 4a, 4b is an electrode pair that combines an ammonium ion selective electrode and a reference electrode, and 5 is a glass bead on which creatininase is immobilized. In the filled enzyme reactor, the buffer solution is sucked by the liquid feed pump 2 and is constantly flowing through the channel at a constant flow rate. The buffer solution is, for example, one in which acetate is dissolved in water to make it alkaline with a pH of 9 or less.

When a certain amount of sample specimen, such as serum, is injected from the sample introduction part 3 using a microsyringe or the like, the selectivity of ammonium ions and other ammonium ions in the sample specimen and buffer solution increases while flowing through the first electrode pair 4a. Detects all the ions of the substance that the electrode senses at once. That is, here, a blank value of the buffer solution containing the sample specimen is detected and supplied to the comparison amplifier 6a together with the output of the comparison electrode which always outputs a constant electromotive force. Note that the ammonium ion selective electrode of electrode pair 4 is formed in a flow-through type and operates with good responsiveness via an electrode membrane.

In the enzyme reactor 5, the reaction of formula (1) is carried out, and the creatinine substrate in the sample is decomposed into N-methylhydantoin and ammonia by the decomposition action of the creatininase enzyme, and the generated ammonia is expressed as (2)
It is converted to ammonium ion in the reaction of Eq. When the decomposed product passes through the ammonium ion selective electrode of the second electrode pair 4b together with the buffer solution, the sum of the ammonium ion and the potential due to the interfering component is detected. This potential is supplied to the comparison amplifier 6b together with the constant potential of the comparison electrode. The reference electrode is, for example, a potassium chloride battery, and is necessary for regulating the ammonium ion selective electrode.

The output of the first electrode pair 4a is supplied to a comparator amplifier 6a.
The output of the second electrode pair 4b is compared with the output of the comparison electrode in a comparison amplifier 6b to obtain a difference signal, which is outputted to a comparison amplifier 6c. Now, in the flow path of FIG. 1, the time when the sample specimen passes through the first electrode pair 4a and the time when the sample specimen passes through the second electrode pair 4b are different. In order to correct this time lag and directly compare the outputs of the comparison amplifiers 6a and 6b, a hold circuit 7 is installed after the comparison amplifier 6a to hold the output signal for a predetermined time, and then the output signals of the comparison amplifiers 6a and 6b are held.
The output of b is input to the comparison amplifier 6c to find the difference.

Now, assuming that the output of the ammonium ion selective electrode of the first electrode pair 4a is S ₁ and the output of the comparison electrode is R ₁ , the output of the amplifier 6a is (S ₁ −R ₁ ).
On the other hand, if the output of the ammonium ion selective electrode of the second electrode pair 4b is _S2 , and the output of the comparison electrode is _R2 , then the output of the comparison amplifier 6b is ( _S2 - _R1 ). Therefore, in the comparator amplifier 6c, (S ₁ −
The difference between R ₁ ) and (S ₂ −R ₁ ) is calculated, but since the comparison electrodes are of the same performance, the value of S ₁ −S ₂ is obtained. That is, the amount of creatinine in the sample that has changed in the enzyme reactor 5 is determined. The output of the comparison amplifier 6c is displayed on a display 8 such as a recorder. FIG. 2 is a recording diagram when a sample is repeatedly introduced, and the height of the peak corresponds to the content of creatinine.

As described above, the creatinine analyzer of this example has a pair of electrodes, each consisting of an ammonium ion selective electrode and a reference electrode, installed in the buffer solution flow path before and after the enzyme reactor in which creatininase enzyme is immobilized. By holding the output from the first electrode pair for a certain period of time and outputting it to the comparison amplifier at the same time as the output from the second electrode pair, creatinine in the sample can be accurately measured without being affected by interference components. This has the effect of allowing analysis to be carried out in detail. Also, the buffer solution is (1)
If the pH value is set to the appropriate value for the reaction in the formula, most of the ammonia in the buffer will be ammonium ions to the extent that it will not interfere with the measurement, so just one type of buffer solution should be constantly circulating. The analysis operation is simple and the equipment can be simplified. Furthermore, since the enzyme is immobilized and can be used for a long time, the amount of enzyme consumed is greatly reduced, which has the advantage of significantly reducing analysis costs.

FIG. 3 is a system diagram of a creatinine analyzer according to another embodiment of the present invention, in which the same parts as in FIG. 1 are given the same reference numerals. In this device, buffer solution tank 1
Three-way Kokko type flow path switching valves 10a and 10b are provided in the flow path between the sample introducer 3 and the ammonium standard solution tank 11 and the substrate standard solution tank 12 to enable communication with the ammonium standard solution tank 11 and the substrate standard solution tank 12. Also, the first point is that a system calibrator 9 is installed between the comparison amplifier 6c and the display 8.
It is different from the illustration.

With this configuration, the entire device can be calibrated, and even if the individual devices are different, the analytical values obtained can be directly compared.
To explain the calibration operation, first, a buffer solution is allowed to flow at a predetermined flow rate, and the flow path switching valve 10a is rotated for a certain short period of time to introduce a certain amount of ammonium standard solution into the flow path from the ammonium standard solution tank 11. After installation, return to the original state. The ammonium standard solution sequentially passes through the first electrode pair 4a and the second electrode pair 4b, and the ammonium ion concentration is detected by the ammonium ion selective electrode. If the outputs of both electrode pairs are equal and the record of the recorder displays a zero value, the performance of both electrode pairs is equal and no calibration is required. However, the above ammonium standard solution must be a sufficiently purified reagent and must not contain creatinine or the like. If the display on the display 8 does not become zero, adjust the adjustment knob of the system calibrator 9 to set it to the zero position.

Next, the flow path switching valve 10b is switched for a short time in the same manner as described above to introduce a certain amount of a solution of creatinine with a known concentration from the substrate standard solution tank 12, and then the original state is returned. Therefore, creatinine is produced in the enzyme reactor 5.
Since ammonium ions are generated by the change in the amount of ammonium ions, a peak as shown in FIG. 2 is recorded on the display 8. If this peak value does not reach a predetermined height proportional to the introduced amount, adjust the sensitivity adjustment knob of the system calibrator 9 so that the predetermined height is displayed. If such sensitivity adjustment can be made continuously, it becomes possible to display a constant value when a certain amount of substrate is introduced even if different devices are used. In addition, if this operation is performed regularly once a day before starting analysis, the capacity of the enzyme reactor 5 can be tested, and the degree of deterioration and consumption of the immobilized enzyme can be determined. This has the advantage that analysis accuracy can be maintained by replacing it in a timely manner.

FIG. 4 is a diagram comparing and showing the relationship between the number of analyzed samples and the analytical values. The broken line 13 indicates a decrease in the analytical value when a large number of samples are analyzed, and this is due to the deterioration of the action due to the flow of the buffer solution of creatininase fixed in the enzyme reactor 5 and the adhesion of impurities. This is thought to be caused by the following. However, when a system calibration means is provided and calibration is performed frequently as in this example, the same analysis value will be shown for a long period of time as shown by solid line 14, and according to the experimental results, 200 samples will be analyzed in this state. I was able to do that. Therefore, the glass beads on which the enzyme is immobilized can be used for a long period of time without being replaced, making it possible to improve analysis efficiency and reduce the cost per sample.

By providing a system calibration means, the creatinine analyzer of this embodiment reduces analysis effectiveness and costs, and also allows direct comparison of data between different devices even if the conditions of the devices are different. This has the effect of making it possible.

In the above embodiment, the hold circuit 7 is used to hold the output difference value of the first electrode pair 4a for a certain period of time, but this may be processed by a small computer using a storage device or the like.

According to the creatinine analyzer of the present invention, continuous analysis is possible and analysis can be performed with less consumption of enzyme amount.

[Brief explanation of the drawing]

Fig. 1 is a system diagram of a creatinine analyzer which is an embodiment of the present invention, Fig. 2 is a recording diagram when a sample is repeatedly introduced, and Fig. 3 is a creatinine analysis device which is another embodiment of the present invention. System diagram of the device, No. 4
The figure is a diagram comparing and showing the relationship between the number of samples to be analyzed and the analysis value. 1...Buffer solution tank, 2...Liquid pump, 3...
Sample introduction part, 4...electrode pair, 5...enzyme reactor,
6... Comparison amplifier, 7... Hold circuit, 8...
Display device, 9...System calibrator, 10...Flow path switching valve, 11...Ammonium standard solution tank, 12
...Substrate standard solution tank.

Claims (1)

[Scope of Claims] 1. A sample introduction section and an enzyme reactor having immobilized creatininase are installed in a flow path for a buffer solution distributed by a liquid pump, and creatinine is introduced from the sample introduction section. In a creatinine analyzer that detects changes in the concentration of ammonium ions in the buffer solution when a sample containing a sample is introduced, ammonium ion selective electrodes are installed in the flow path before and after the enzyme reactor, and ammonium ion selective electrodes are installed in the flow path before and after the enzyme reactor. a selective electrode and a comparison electrode are provided together to form a first electrode pair and a second electrode pair;
In order to obtain the output signal difference when the sample specimen passes through the first and second electrode pairs, the signal value from the first electrode pair is used as a signal until the sample specimen passes through the second electrode pair. A creatinine analyzer characterized in that it is configured to be held in a holding means and to detect a difference in concentration of ammonium ions in the buffer solution. 2. The creatinine analyzer according to claim 1, wherein the signal holding means is a means for causing a hold circuit to hold the output signal of the first electrode pair. 3. The buffer solution flow path includes two channels that can independently introduce the ammonium ion standard solution with a known concentration and the creatinine standard solution with a known concentration into the flow path upstream of the first electrode pair. The creatinine analyzer according to claim 1, wherein the creatinine analyzer is a flow path provided with a flow path switching valve.