JPH04166099A - Method for determining glucose - Google Patents

Abstract

PURPOSE:To stably carry out determination of glucose over a long period by adding a specific alkyl alcohol to an solution to be examined at a specific amount when change of a substance by enzymatic reaction of glucose is measured using electrodes. CONSTITUTION:When glucose is determined using electrodes for measuring change of substance by enzymatic reaction of glucose, 0.01-1wt.% 6-8C alkyl alcohol (preferably n-hexyl alcohol) is added to a solution to be examined. A glucose oxidase is used as an enzyme catalyzing reaction of the glucose and simultaneously oxygen electrodes capable of measuring an amount of oxygen reduced or H2O2 electrodes capable of measuring an amount of H2O2 produced are preferably used as the electrodes.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for quantifying glucose using an enzymatic reaction.

(Prior Art and its Problems) Various methods are known for quantifying glucose by enzymatic reaction. For example, hydrogen peroxide generated by an enzymatic reaction involving glucose oxidase is mixed with peroxidase and 0-
There is a method of colorimetric determination using a coloring reagent such as toluidine, and a method of quantifying the reduction of nicotinamide adenine dinucleotide by ultraviolet absorption in a reaction in the coexistence of hexokinase and glucose 6-phosphate dehydrogenase. . Also,
Examples of methods using electrodes include a method in which dissolved oxygen reduced by an enzyme reaction involving glucose oxidase is measured using an oxygen electrode, and a method in which generated hydrogen peroxide is measured using a hydrogen peroxide electrode. Glucose oxidase, which catalyzes the enzyme reaction, can be added in the form of a solution or repeatedly used as an immobilized enzyme.

In any case, the method using this electrode is easy to mechanize and is suitably employed in automatic analyzers, including data processing by computer.

However, the method using this electrode has the problem that when measured over a long period of time, it gradually shows an abnormal value and eventually becomes impossible to measure.

c) The present inventors have made extensive research in order to solve the above problem.
The present inventors have discovered that by having a specific amount of alkyl alcohol present, the product can be used for a long period of time without exhibiting any abnormal values, and the present invention has been completed.

That is, the present invention provides a method for quantifying glucose using an electrode that measures the substance change of glucose due to an enzyme reaction.
] This is a method for quantifying glucose, characterized in that the glucose is present in an amount of 1 to 11ti-%.

Known enzymes that catalyze the glucose reaction can be used. For example, glucose oxidase, glucose dehydrogenase.

Hexokinase, etc. Among these, glucose oxidase catalyzes the reaction between glucose and oxygen to produce gluconic acid and hydrogen peroxide, and is therefore particularly suitable for use with an electrode that detects oxygen or hydrogen peroxide. The enzyme needs to be brought into contact with the analyte solution in order to catalyze the glucose reaction, and there are two methods: adding the enzyme separately to the analyte solution and making an immobilized enzyme exist in the analyte solution.

A method using an immobilized enzyme is particularly suitable industrially because the enzyme can be used repeatedly. The method for immobilizing the enzyme is not particularly limited, and known methods such as a method of covalently bonding the enzyme to a porous membrane or a method of immobilizing the enzyme within the membrane by crosslinking are employed.

The analyte solution is composed of a analyte solution containing glucose and water, preferably a buffer solution, and an enzyme is present in the analyte solution to carry out the desired enzymatic reaction. The test solution and buffer may be mixed in advance, or may be mixed after being separately placed in a reaction vessel containing the enzyme.

The test liquid may be any type of liquid as long as it contains glucose and can be measured by an enzyme reaction. For example, human or animal blood.

Examples include serum, urine, and fermentation liquids and culture media used for fermentation of various foods and feeds, which are diluted as necessary before being subjected to measurement.

The buffer is used to stably perform the enzymatic reaction, and any buffer that does not inhibit the enzymatic reaction can be used without particular limitation.

Examples of buffers that are generally suitably used include Haba, phosphate buffer, Tris buffer, and citrate buffer. In the buffer, H is selected within a range that provides a sufficiently high enzymatic activity of the enzyme used. In particular, in the case of glucose oxidase, a pH of about 5 to 7 is suitable. The reaction temperature is preferably constant, and generally a specific temperature selected in the range of 20 to 50°C is used. Particularly in the case of glucose oxidase, selection in the range of 30 to 40°C is suitable.

Any known electrode can be used as the electrode for measuring the change in substance caused by the enzymatic reaction, as long as the change in substance can be measured. Examples include an oxygen electrode that measures the amount of oxygen decreased due to the enzymatic reaction described above, and a hydrogen peroxide electrode that similarly measures the amount of hydrogen peroxide produced. Generally, the electrode is installed in the enzyme reaction cell and the electrode is used to measure the enzyme reaction at the same time, but the analyte solution before and after the enzyme reaction may be measured separately using the electrode. Furthermore, a configuration in which the above-mentioned immobilized enzyme is placed on an electrode and substance changes caused by the enzyme reaction are detected simultaneously and with higher sensitivity is also suitably used.

As an apparatus for carrying out the measuring method of the present invention, a commercially available apparatus can be used as it is or with some modification. An example of the device is shown in FIG. Buffer solution 1 is sent to enzyme reaction cell 6 by buffer solution syringe 2 . A fixed amount of the test liquid 4 is sucked in by the sample probe 5, moved to the enzyme reaction cell 3, and discharged into the cell.

The enzyme reaction cell is equipped with a stirring bar 6 and a stirring motor 7 to mix the test liquid and the buffer solution.

An oxygen electrode 8 equipped with a membrane immobilized with glucose oxidase is attached to the enzyme reaction cell, and its output is converted to a glucose concentration by a microprocessor 10 via an amplifier 9, and the value is displayed on a display I/I 11. 3. From buffer solution 1 to enzyme reaction cell 3. The oxygen electrode 8 portion is kept at a constant temperature in a constant temperature bath 12. The liquid after the reaction is discharged through a waste liquid line 15.

The greatest feature of the present invention is that an alkyl alcohol having 6 to 8 carbon atoms is present in the sample solution when glucose is determined.

The alkyl alcohol used in the present invention has 6 to 8 carbon atoms.
As the alkyl alcohol, known linear or branched alkyl alcohols can be suitably used. Specific examples of alkyl alcohols that are particularly effective in the present invention include n-alkyl alcohols having 6 to 8 carbon atoms, such as n-hexyl alcohol, n-heptyl alcohol, and n-octyl alcohol. Alkyl alcohols with less than 5 carbon atoms are less effective.
A higher concentration is required to exhibit an effect equivalent to that of an alkyl alcohol having 6 or more carbon atoms. Addition of such a large amount of alcohol is not preferable because it reduces enzyme activity.

On the other hand, alkyl alcohols having 9 or more carbon atoms are not effective because they do not dissolve in water.

The amount of the alkyl alcohol added is 0.
It is preferably used in a range of 0.01 to 1% by weight. 0.01
If the amount is less than 1% by weight, the effect will not be sufficient, and if it exceeds 1% by weight, it will be difficult to dissolve uniformly in the analyte solution and enzyme activity will decrease, which is undesirable.

There are no particular limitations on the mode in which the alkyl alcohol is present in the analyte solution in the present invention, and the alkyl alcohol can also be added to the analyte solution during the enzyme reaction.

However, it is generally preferable to pre-contain a certain concentration of alkyl alcohol in the test solution and/or buffer solution.

(Effects) By employing the present invention, the glucose concentration in the test liquid can be stably quantified over a long period of time. In addition, the cost of replacing electrodes, cleaning the device, etc. can be streamlined, and measurement costs can be reduced.

However, the mechanism by which the effects of the present invention are brought about is not clear. Although it is generally known that alcohols have a bactericidal effect, the fact that the alkyl alcohol having 6 to 8 carbon atoms used in the present invention does not bring about the effects of the present invention solely due to its bactericidal effect, for example, This can be inferred from the fact that the effects of the present invention cannot be obtained even if formalin, which is known to have the same content, is used in place of the alkyl alcohol of the present invention. Based on this, the present inventors have discovered that specific alkyl alcohols have the effect of inactivating various organic substances contained in the test solution or buffer solution, so that the effect that interferes with quantitative determination is suppressed, and as a result, long-term We estimate that it will be possible to quantify over a wide range of areas.

(Examples) In order to explain the present invention in more detail, Examples and Comparative Examples will be described below, but the present invention is not limited to these Examples.

Example 1 The apparatus used was Glucoloder-E manufactured by Analytical Instruments (using an oxygen electrode equipped with a membrane on which glucose oxidase was immobilized). Phosphate buffer (0.1M, pH=6.9) was used as the buffer solution, and used for the measurement as it was or with 0.1% by weight of n-hexyl alcohol added thereto. 50 μt of horse serum (manufactured by Nippon Biotest Co., Ltd.) was used as a sample solution, and the glucose content was measured by injecting it into an enzyme reaction cell together with a 1.8-mL buffer solution. 10
Each sample was calibrated with a glucose standard solution of 0 ray/ltt and 200/111. The apparatus was operated continuously for 10 hours each day. The results are shown in Table 1.

Table 1 Example 2 In the same manner as in Example 1, the alkyl alcohols shown in Table 2 were added to the buffer solution and measured. The results are shown in Table 2. , Example 3 The apparatus used in Example 1 was modified and a membrane on which glucose oxidase was immobilized was attached to the electrode. Measurements were carried out in the same manner as in Example 1, except that a hydrogen peroxide electrode was used. The results are shown in Table 3.

Table 3 Comparison f River 1 Buffer,! = 1. Example 1 except that 0.1 iE amount % of formalin was used instead of -n-hexyl alcohol.
Measure 1- in the same manner as . The results are shown in Table 4.

Table 4 Comparative Example 2 Measurement was carried out in the same manner as in Example 1 except that the concentration of n-hexyl alcohol was changed. The results are shown in Table 5.

Table 5

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of an apparatus for analyzing glucose. 1 buffer solution, 2 syringe for buffer solution, 3 enzyme reaction cell, 4 test solution, 5 probe for sample. 6 stirring bars, 7 stirring motors, 8 oxygen electrodes equipped with enzyme-immobilized membranes, 9 amplifiers. 10 microprocessors, 11 displays. 12 constant temperature bath, 13 waste liquid line

Claims (1)

[Claims] Glucose characterized by the presence of 0.01 to 1% by weight of an alkyl alcohol having 6 to 8 carbon atoms in a sample solution when quantifying glucose using an electrode that measures substance change due to an enzymatic reaction of glucose. Quantification method.