JPH10142193A - Method and apparatus for measuring concentration with utilizing phchange reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simpl and stably measure a concentration of each substance related to a biological reaction, a biochemical reaction or a chemical reaction with the use of common electrodes and apparatus without using special electrodes and apparatus for each substance. SOLUTION: A sample solution including a substance to be measured is borough into contact with a film 7 or column holding a labile substance which generates a pH change through a biological, biochemical or chemical reaction with the substance to be measured. A potential difference of two pH-sensing electrodes 5, 6 soaked in the sample solution before and after the touch in the liquid junction state is measured. The obtained potential difference is compared with a potential difference preliminary measured for a solution including the substance to be measured of a known concentration. A concentration of the substance to be measured in the sample solution is obtained in the method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring the concentration of a substance contributing to each of these reactions by utilizing a pH change caused by a biological, biochemical or chemical reaction, and a method for measuring the same. Device.

[0002]

2. Description of the Related Art Conventionally, biochemical oxygen consumption (BOD), which is an indicator of water pollution, has been measured using a microorganism sensor having fungi immobilized thereon. In the fields of medicine and food, an enzyme sensor using an enzyme (glucose oxidase) is used for measuring glucose concentration.

For example, in the measurement of glucose, a substance produced by a biochemical reaction in which glucose oxidase oxidizes glucose to gluconic acid is measured using an immobilized enzyme membrane or an immobilized enzyme column on which glucose oxidase is immobilized. Was. Specifically, the glucose concentration is determined by measuring the oxidation current of hydrogen peroxide generated by the reaction or by measuring the reduction current of oxygen consumed by the reaction.

[0004] However, in these measuring methods, a substance produced by a biological reaction or a biochemical reaction is used.
For measurement using an electrode selectively sensitive to the substance, for example, an oxygen electrode, a hydrogen peroxide electrode, an ammonia gas electrode, an ammonia ion electrode, a carbon dioxide gas electrode, etc., a dedicated electrode and a measuring device for each substance to be generated Had to be used. In addition, when a column is used for immobilizing an enzyme or the like, the sensitivity is apt to decrease, so that the measurable substances and concentrations are limited.

[0005] Some of the measuring methods utilizing these biological and biochemical reactions are carried out by ordinary pH measurement. Since the pH measurement is performed using the electrode and the reference electrode, the influence of a temperature change becomes large, and constant temperature control or constant temperature control is required. In addition, the maintenance and inspection of the comparative electrode is very troublesome because the internal liquid needs replenishment and replacement.

[0006]

SUMMARY OF THE INVENTION In view of the above-mentioned circumstances, the present invention provides a method for controlling the concentration of a substance involved in a biological reaction with a fungus, a biochemical reaction with an enzyme, or a chemical reaction.
A method that does not require a dedicated electrode or device for each substance to be measured, uses a common electrode or device, and can easily and stably measure even if the concentration of the substance to be measured is low, and It is an object of the present invention to provide a measuring device therefor.

[0007]

The present invention focuses on the fact that in various reactions including biological reactions and biochemical reactions, the pH in a solution often fluctuates due to a change in a substance due to the reaction. The variation of pH is common p regardless of the type of reaction.
The measurement is performed using an H-sensitive electrode to determine the concentration of the substance to be measured involved in the reaction.

That is, in the method of the present invention, a sample liquid containing a substance to be measured is brought into contact with a reactive substance which causes a pH change by a biological, biochemical or chemical reaction with the substance to be measured, The potential difference between the two pH-sensitive electrodes immersed in the sample solution before and after the contact in the state is measured, and the obtained potential difference is compared with a potential difference measured in advance for a solution containing a substance to be measured having a known concentration. It is characterized in that the concentration of a substance to be measured in a sample liquid is obtained.

In order to carry out the concentration measuring method utilizing the pH fluctuation reaction, the measuring device of the present invention comprises two pH-sensitive electrodes arranged along a liquid flow path, and two pH-sensitive electrodes disposed in the liquid flow path.
Placed between the sensitive electrodes,
It is provided with a reaction active substance that causes a pH change due to a biochemical or chemical reaction, and a potential difference detecting unit that detects a potential difference between the two pH-sensitive electrodes.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, the use of a reaction-active substance that causes a pH fluctuation due to a reaction with a substance to be measured allows the pH fluctuation before and after each reaction to be reduced by using only two pH-sensitive electrodes. It is used to measure the potential difference between the electrodes. Therefore, a common pH-sensitive electrode can be used for many biological, biochemical, and chemical reactions, and the concentration of the target substance can be easily and stably measured without being affected by temperature changes. It is possible.

The substance to be measured may be any substance that produces or consumes an acidic or alkaline substance by various reactions with a reaction active substance, thereby causing a pH fluctuation. Accordingly, the reaction active substance may be any substance that causes the above-mentioned pH fluctuation with the substance to be measured, and includes not only fungi and enzymes, but also general chemical substances and ion exchange resins. .

For example, when the substance to be measured is glucose, if glucose oxidase is used as a reaction active substance, the pH changes to an acidic side due to gluconic acid generated by a biochemical reaction. When the substance to be measured is urea, ammonia is generated by a biochemical reaction with the reactive substance uriase, so that the pH shifts to an alkaline side. When the substance to be measured is glutamic acid, D
-Glutaminase is used as a reactive substance, and ammonia is similarly generated to cause a pH change.

The biological reaction includes a reaction in which nitric acid bacteria as a reaction active substance metabolize ammonia or nitrous acid to nitric acid, and a reaction in which lactic acid bacteria decompose lactose and metabolize it to lactic acid. Shifts to the acidic side before and after the reaction. Further, examples of chemical reactions that cause pH fluctuation include:
There are reactions in which the H ⁺ type ion exchange resin adsorbs metal ions and the like and releases hydrogen ions, and reactions in which the complexing agent forms a complex with the metal ions and releases hydrogen ions.

The reactive substance is located between the two pH-sensitive electrodes. A preferred method for this is to attach a membrane holding or fixing a reactive substance to the surface of the sensitive part of the pH-sensitive electrode disposed downstream of the liquid flow path, or to use a liquid flow between the two pH-sensitive electrodes. There is a method of holding or immobilizing a reactive substance on a column or a membrane provided in the middle of a path.
In addition, a method for holding or immobilizing a reactive substance on a membrane or a column is already known, and for example, a covalent bonding method, an adsorption method, a cross-linking method, etc. are known for enzymes.

Further, in the present invention, the potential difference between each pH-sensitive electrode is measured using two pH-sensitive electrodes regardless of the substance to be measured. If the liquid junction between the two pH-sensitive electrodes is in the sample liquid state, the potential difference between the pH-sensitive electrodes can be measured without using a reference electrode. As such a pH-sensitive electrode, besides a normal glass electrode, there is an electrode having a metal oxide such as iridium oxide or tantalum oxide that is sensitive to hydrogen ions as a sensitive part, and these electrodes are used alone (without using a comparative electrode). What is necessary is just to use combining two. Therefore, since no reference electrode is used, maintenance and inspection are easy, and stable measurement can be performed without being affected by temperature.

In the method of the present invention, first, a measuring device including two pH-sensitive electrodes is calibrated. For example, a downstream side where a first pH-sensitive electrode and a second pH-sensitive electrode and a reactive substance, or a membrane holding or immobilizing the first pH-sensitive electrode and the reactive substance are attached to a standard solution composed of a phosphate buffer. Is immersed, and the potential difference between the first and second pH-sensitive electrodes at this time is corrected to zero.

Next, a sample liquid containing the substance to be measured is continuously supplied to the liquid flow path, and flows from the first pH-sensitive electrode to the second pH-sensitive electrode via the reactive substance. It is necessary to adjust the pH of the sample solution with a phosphate buffer so as to be almost the same as the standard solution at the time of calibration.

Since the substance to be measured in the sample solution releases or consumes protons, acidic substances, or alkaline substances by reacting with the reactive substance, the pH of the sample liquid on the downstream second pH-sensitive electrode side decreases. fluctuate. Accordingly, the potential difference between the first and second pH-sensitive electrodes at this time is measured, and the obtained potential difference is compared with a potential difference previously measured for a solution containing a substance to be measured having a known concentration, whereby the potential difference in the sample solution is measured. The concentration of the substance to be measured can be determined.

According to the present invention, the range of substances to be measured is wider than before, and almost all substances that cause pH fluctuation by biological, biochemical or chemical reactions can be measured. In addition, since the effects of temperature changes and changes in the base solution that occur during measurement can be eliminated by using multiple pH-sensitive electrodes, stable measurements can be easily performed without considering these effects. Become.

[0020]

EXAMPLE 1 As shown in FIG. 1, a first cell 1 and a second cell 2 were
And a pump 4 was installed downstream of the second cell 2 in the liquid flow path 3. The first cell 1 has a sensitive part 5 of iridium oxide.
A first pH-sensitive electrode 5 having a was attached. Also, the second cell 2 was provided with the second pH-sensitive electrode 6 having the iridium oxide sensitive portion 6a.
Enzyme membrane 7 of glucose oxidase so as to cover
Was fixed with the membrane retainer 6b.

A phosphate buffer solution containing no glucose is continuously supplied to the liquid flow path 3, and the first pH-sensitive electrode 5 and the second pH
The potential difference between the sensitive electrodes 6 was calibrated to zero. Next, a sample solution containing glucose was continuously supplied to the liquid flow path 3, and a potential difference between the first pH-sensitive electrode 5 and the second pH-sensitive electrode 6 was detected by a potential difference detecting unit of the device.

At this time, glucose in the sample solution reacts with glucose oxidase of the immobilized enzyme membrane 7 to become gluconic acid, and shifts the pH to the acidic side. Therefore, the pH of the sample solution before the reaction is detected by the first pH-sensitive electrode 5 and the pH of the sample solution containing gluconic acid after the reaction is detected by the second pH-sensitive electrode 6. The potential difference between them is measured.

In this way, for a plurality of sample solutions having different glucose concentrations, the first and second pH-sensitive electrodes 5,
When the potential differences between the samples Nos. 6 were measured, the results shown in FIG. 3 were obtained. FIG. 3 shows a calibration curve, and excellent linearity was obtained in a range of glucose concentration up to 200 ppm.

Therefore, when the potential difference between the first and second pH-sensitive electrodes 5 and 6 is measured for the sample solution containing glucose of unknown concentration by the same operation as described above, the obtained potential difference is shown in FIG. The glucose concentration in the sample solution can be determined using the calibration curve.

Example 2 As shown in FIG. 2, the immobilized enzyme membrane 7 of Example 1 (FIG. 1)
Instead of this, an immobilized enzyme column 8 holding glucose oxidase was used, and this immobilized enzyme column 8 was attached in the middle of the liquid flow path 3 between the first cell 1 and the second cell 2. Other parts of the device are the same as in the first embodiment.

After performing calibration using this measuring device in the same manner as in Example 1, a sample solution containing glucose having a changed concentration is continuously supplied to the liquid flow path 3, and the first and second pH values are measured. When the potential difference between the sensitive electrodes 5 and 6 was measured, the result shown in FIG. 4 was obtained. Although the measurement range was narrower than that of Example 1, excellent linearity was obtained in a range up to a glucose concentration of 100 ppm.

Example 3 BOD immobilized with yeast and Bacillus subtilis instead of the enzyme membrane immobilized with glucose oxidase used in Example 1 above.
The glucose concentration in the sample solution was measured in the same manner as in Example 1 except that the sensor membrane was attached to the second pH-sensitive electrode.

Microorganisms in the BOD sensor membrane decompose glucose in the sample solution and simultaneously consume oxygen in the sample solution to release carbon dioxide. The amount of released carbon dioxide is proportional to the amount of glucose decomposed by the microorganism, and the released carbon dioxide dissolves in the sample solution to become carbonic acid, and shifts the pH to the acidic side. Therefore, the glucose concentration in the sample solution can be determined by measuring the potential difference between the first pH-sensitive electrode not in contact with the BOD membrane and the second pH-sensitive electrode in contact with the BOD membrane.

In this example, when the potential difference between the first and second pH-sensitive electrodes was measured, the result shown in FIG. 5 was obtained. By using FIG. 5 as a calibration curve, it is possible to determine the glucose concentration of the sample solution of unknown concentration using the same BOD sensor membrane and pH-sensitive electrode. In addition, since the same applies to other nutrients in addition to glucose, this measuring device can also function as a nutrient measuring device (BOD measuring device).

[0030]

According to the present invention, the concentration of a substance involved in a biological reaction, a biochemical reaction, or a chemical reaction can be adjusted without using a dedicated electrode or device for each substance to be measured. It is possible to easily and stably perform measurement using a device including two common pH-sensitive electrodes, and even when the substance to be measured has a low concentration.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a specific example of a concentration measuring device of the present invention.

FIG. 2 is a schematic view showing another specific example of the concentration measuring device of the present invention.

FIG. 3 is a graph showing the relationship between the glucose concentration and the potential difference obtained in Example 1.

FIG. 4 is a graph showing a relationship between a glucose concentration and a potential difference obtained in Example 2.

FIG. 5 is a graph showing the relationship between the glucose concentration and the potential difference obtained in Example 3.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 1st cell 2 2nd cell 3 Liquid flow path 4 Pump 5 1st pH sensitive electrode 5a Sensitive part 6 Second pH sensitive electrode 6a Sensitive part 6b Membrane holding 7 Immobilized enzyme membrane 8 Immobilized enzyme column

Claims (4)

[Claims] 1. A sample solution containing a substance to be measured is brought into contact with a reactive substance which causes a pH change by a biological, biochemical or chemical reaction with the substance to be measured, and before and after the contact in a liquid junction state The potential difference between the two pH-sensitive electrodes immersed in the sample solution is measured, and the obtained potential difference is compared with a potential difference previously measured for a solution containing a substance to be measured having a known concentration, and the measurement in the sample solution is performed. A concentration measuring method using a pH fluctuation reaction, wherein a concentration of a target substance is obtained. 2. A method according to claim 1, wherein the two pH-sensitive electrodes are arranged along the liquid flow path, and the two pH-sensitive electrodes of the liquid flow path are arranged between the two pH-sensitive electrodes to form a biological or biochemical reaction with the substance to be measured. Or a reaction-active substance causing a pH change by a chemical reaction;
A concentration measuring device using a pH fluctuation reaction, comprising: a potential difference detecting section for detecting a potential difference between the pH-sensitive electrodes. 3. The p according to claim 2, wherein the reactive substance is held by a membrane attached to the surface of a sensitive part of a pH-sensitive electrode disposed downstream of the liquid flow path.
Concentration measuring device utilizing H fluctuation reaction. 4. The pH fluctuation according to claim 2, wherein the reactive substance is held in a column or a membrane provided in the middle of a liquid flow path between two pH-sensitive electrodes. A concentration measuring device that uses a reaction.