JPS6214780B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to a multi-item simultaneous measurement device,
In detail, in a multi-item simultaneous measurement device that simultaneously measures multiple components in a test solution in a reaction tank using enzyme electrodes, each enzyme electrode is equipped with an immobilized enzyme of a different type of enzyme. Multiple working electrodes and one
An enzyme electrode consisting of two counter electrodes, which can apply an applied voltage to each working electrode, and a voltage applying device for applying an applied voltage is disposed at both ends of the working electrode and the counter electrode. This invention relates to a simultaneous measurement device.

A multi-item simultaneous measurement device is used to measure the amount of substances contained in biological fluids such as blood and urine, culture fluids, fermentation fluids, foods, etc. used in manufacturing and testing in the pharmaceutical, medical, and food fields. This device can measure the concentration of multiple components simultaneously. This multi-item simultaneous measurement device uses multiple enzyme electrodes each equipped with different immobilized enzymes.
Each enzyme electrode is placed in a single measurement container, and a test solution is added to the container to simultaneously measure the concentration of each component in the test solution on which the enzymes act. (Special application 1973-
No. 118447 (Japanese Unexamined Patent Application Publication No. 1983-44954), Patent Application No. 118447-
No. 52717 (Japanese Unexamined Patent Publication No. 144537, 1982), Patent Application No. 144537
No. 140449 (Japanese Unexamined Patent Publication No. 56-64653).

By the way, as each electrode used in these multi-item simultaneous measurement devices, an electrode with a working electrode and a counter electrode is used as a pair, and each enzyme electrode is equipped with each immobilized enzyme in the detection part of the electrode. It was hot.
Therefore, one electrode is required for each item to be measured, and when these multiple electrodes are arranged in one container, the volume of the measurement container is , which required more volume than necessary. Moreover, for this reason, the amount of test liquid used was also larger than necessary.

The object of the present invention is to provide a multi-item simultaneous measurement device that can be installed in a smaller container by improving the structure of the plurality of electrodes. Therefore, it is particularly effective in cases where only a small volume of test fluid can be collected, for example, in clinical diagnostic analyzes using biological fluids.

According to the present invention, an electrode composed of a plurality of working electrodes and one counter electrode is used, and each working electrode is provided with an immobilized enzyme of a different type, and when it comes into contact with a test liquid, The concentration of components produced by the enzymatic reaction of the immobilized enzyme, such as hydrogen peroxide, and components consumed, such as oxygen, are measured. In addition, during measurement, the values of multiple components to be measured are obtained as electrical changes by applying an applied voltage to both ends of the working electrode and counter electrode, which can be applied an applied voltage to each working electrode. .

Next, a specific example of the present invention in which hydrogen peroxide, which is one of the components produced using oxidase, is measured will be explained with reference to the drawings.

Ultra-fine (for example, usually 0.3 to 3 mm) embedded in an insulator 12 such as glass or Teflon (registered trademark)
The tip of the working electrode 4 is made of a platinum wire (in the case of Teflon, it is coated with a platinum wire to a thickness of several microns), and the tip working part is fixed. It is equipped with enzyme 6. This working electrode 4 is connected to a measurement container (usually 0.1~
(approximately 0.5 ml) 3, so that their action parts come into contact with the test liquid in the reaction tank 7. At this time, it is preferable that the plurality of working electrodes 4 are arranged radially on the side wall of the reaction tank 7 in the measurement container 3. On the other hand, as the counter electrode 8, an electrode material made of a silver film may be disposed inside the reaction tank 7, and generally the bottom wall thereof constitutes a part of the reaction tank 7. In addition, platinum, silver, rhodium, etc. are preferably mentioned as the above-mentioned electrode material, and the counter electrode 8 is usually the working electrode 4.
It is sufficient if the area ratio is approximately 100 times or more. In addition, each working electrode 4 and counter electrode 8
The output is taken out by each lead wire 9, 10, and furthermore, this output is taken out from the electrodes 4 and 8, in the case where there are four working electrodes as shown in FIG. 16 (16-1, 16-
2, 16-3, 16-4), each working electrode 4
A voltage application device V is connected to both ends of the A side of the side and the B end of the counter electrode 8 side, and a microammeter 14 (for example, 14-
1, 14-2, 14-3, 14-4), variable resistor 15 (for example, 15-1, 15-2, 15-
3, 15-4) to read the microammeter value, or record the electrical change in the variable resistor by connecting it to the detection site of the recorder. In addition, at this time, the voltage applying device V may be one for applying a load between each working electrode 4 and the counter electrode 8,
It is usually about 0.5 to 1 V, preferably about 0.7 V, and if it is set to cathode potential, it will detect not only hydrogen peroxide but also dissolved oxygen, so an operation to reduce the amount of dissolved oxygen is required. Only sensing anodic potential loads are preferred. Further, the voltage applying device V used may be provided at each end of each working electrode 4 and counter electrode 8, but preferably the voltage applying device V is
It is sufficient to apply a load to both ends of the end portion and the B end portion using one voltage application device.

Furthermore, generally, an inlet tube 2 for a buffer solution and an inlet tube 1 for a test liquid such as a microsyringe or a micropipette are arranged above the measurement container 3, and a discharge tube for discharging the liquid from the bottom. 11 will be provided.

With this device, multiple components can be measured simultaneously, either batchwise or continuously.

Furthermore, when performing multi-item simultaneous measurement using such an apparatus, the following operations are performed.

First, a buffer solution is added to the reaction tank 7 in the measurement container 3, and a test solution is added while being stirred by the stirrer 13. By adding the test solution, a reaction occurs in the enzyme immobilized on each working electrode 4, and as a result, hydrogen peroxide is produced. Also, regarding this reaction,
Preferably, 5 seconds to 1 minute at 37°C is sufficient. After the reaction is completed, the test liquid etc. are discharged, a buffer solution is added for washing, and the next reaction is repeated. In addition, the hydrogen peroxide produced by the reaction is electrically changed by applying a voltage to both ends of the working electrode and the counter electrode with a voltage applying device.
Find the microammeter value or the electrical change value of the variable resistor.

By measuring hydrogen peroxide in this way,
The multidimensional value after the reaction is exactly the same level as the level before use in each measurement, and the measurement time is quick, making it possible to measure extremely accurately.

It is also clear that these operations can be performed manually or automatically.

In this way, the multi-item simultaneous measurement device of the present invention
Because of the improved electrodes, the volume of the reaction vessel can be significantly reduced, which is particularly advantageous when measuring multiple biological fluids simultaneously. Moreover, although the above-mentioned specific example illustrates the measurement in a container, the electrode may be arranged in a reaction tank such as a pipe through which the test liquid is continuously flowing.

Examples of test liquids that can be used include blood such as serum and plasma, biological fluids such as urine, fermentation liquids, culture liquids, food products, and the like. In addition, examples of the components to be measured in these test liquids include glucose, lactic acid, cholesterol, cholesterol ester, triglyceride, phospholipid, glycerol, uric acid, pyruvic acid, creatinine, and creatine. Furthermore, as the enzyme used as the immobilized enzyme, oxidase which uses the component to be measured as a substrate is usually used. For example, lactate oxidase is used for lactic acid, cholesterol oxidase is used for cholesterol, and cholesterol esterase and cholesterol are used for cholesterol ester. Oxidase, lipase or lipoprotein lipase and glycerol oxidase for triglycerides, or glycerokinase, ATP, L-α-glycerophosphate oxidase, phospholipase D or phospholipase C for phospholipids, alkaline phosphatase and choline oxidase, glycerol if glycerol oxidase, or glycerokinase,
ATP, L-α-glycerophosphate oxidase,
Various enzymes are selected and used, such as uricase for uric acid, pyruvate oxidase for pyruvate, creatininase, creatinase and sarcosine oxidase for creatinine. Furthermore, these enzymes are optionally entrapping immobilized with polyacryloamide and mixed with insoluble proteins.
Various types of immobilization such as cross-linking, immobilization by entrapping or cross-linking with collagen, fibroin, etc., immobilization by adsorption or cross-linking with porous polymer resin, entrapping immobilization with photocurable resin, etc. It is immobilized by enzyme means and is used in the form of a membrane or fiber.

Furthermore, instead of detecting hydrogen peroxide as described above, it may be carried out using oxygen, that is, the amount of oxygen, which is one of the components consumed in the enzyme reaction, may be electrically changed at the electrode. By performing the same procedure below, simultaneous measurement of multiple items can be achieved.

[Brief explanation of the drawing]

FIG. 1 shows an embodiment of the present invention, and in the figure, 1 is an introduction tube for the test liquid, 2 is an introduction tube for the buffer solution,
3 is a measurement container, 4 is a working electrode, 5 is a platinum wire, 6 is an immobilized enzyme, 7 is inside a reaction tank, 8 is a counter electrode, 9 is a lead wire, 10 is a lead wire, 11 is a discharge pipe, 12 is an insulator , 13 shows a stirrer, FIG. 2 shows a measuring system based on an electrical change based on an embodiment of the present invention, 14 shows a microammeter, 14-1,
14-2, 14-3, 14-4 are each microammeter, 15 is a variable resistor, 15-1, 15-
2, 15-3, 15-4 are each variable resistor, 16 is an electrode, 16-1, 16-2, 16-3, 16-4
are each electrode indicating each working electrode and counter electrode, A is the end from the working electrode side, B is the end from the counter electrode side, and V is a voltage applying device for applying voltage to the A end and the B end. show.

Claims (1)

[Claims] 1. In a multi-item simultaneous measurement device that simultaneously measures multiple components in a test solution in a reaction tank using enzyme electrodes, each enzyme electrode measures different types of enzymes individually. Multiple working electrodes with immobilized enzymes and 1
An enzyme electrode consisting of two counter electrodes, which can apply an applied voltage to each working electrode, and a voltage applying device for applying an applied voltage is disposed at both ends of the working electrode and the counter electrode. Simultaneous measurement device. 2. The multi-item simultaneous measurement device according to claim 1, wherein the working electrode is a working electrode that measures the concentration of hydrogen peroxide produced by an enzyme reaction. 3. The multi-item simultaneous measurement device according to claim 1 or 2, which is a reaction tank in which the counter electrode constitutes a part of the reaction tank and the working electrode is arranged in the reaction tank.