JPS6147199A - Method of analysis using enzymatic reaction - Google Patents

Abstract

PURPOSE:To prolong life of an enzyme, to prevent occurrence of foam, and to analyze a substrate in a diluted solution up to a high concentration in high accuracy, by feeding diluted solution which is passed through a pipe having permeability to air in a constant temperature bath kept at a specific temperature to a detector. CONSTITUTION:A diluted solution such as an aqueous solution of a phosphate, etc. in the container 9 is passed through the temperature controlling coil 6 consisting of a pipe, such as silicon rubber, etc. having permeability to air set in the constant temperature bath 5 kept at <=25 deg.C lower than room temperature by the pump 8 so that the temperature in the diluted solution is made constant, dissolved oxygen in the diluted solution is saturated, a solution containing a substrate such as beta-D glucose, etc. is added by the sample injector 7 to the diluted solution, which is introduced through the mixing coil 6' to the flow cell 2, and stirred by the stirrer 4 by rotating the rotor 3. Concentration of hydrogen peroxide which is generated a given time after being introduced is taken out as a peak electric potentially by the enzymatic electrode 1 such as glucose oxidase 50mU to which glucose oxidase is attached, and concentration of a substrate such as glucose, etc, is calculated through the amplifier 12 and the microcomputer 13.

Description

[Detailed Description of the Invention] [Industrial Application Field] Quantitative analysis of natural substances etc. using enzymatic reactions is
It has high selectivity due to its substrate specificity, and can be used at normal pressure and mild temperature.
Since it can be measured at a pH close to neutrality, it is used in the fields of food analysis and clinical analysis. Analytical methods using enzymes basically include end-point detection methods, which carry out measurements after the enzymatic reaction has completely completed, and reaction rate detection methods, which take advantage of the equilibrium of the enzymatic reaction and carry out measurements mid-way through the reaction. . The present invention relates to the latter.

[Conventional technology]

When performing an analysis using an enzymatic reaction, it is necessary to precisely control the reaction time, temperature, etc. in order to keep the analysis conditions constant.Enzymatic reactions are highly temperature dependent, so temperature control is particularly important. is important.

Conventionally, analysis has been carried out at a constant temperature in the vicinity of 30 to 37 C, which is higher than room temperature, because of the need to perform analysis quickly and to bring the measurement temperature close to the body temperature in clinical chemistry tests. When performing analysis at such temperatures, firstly, the high temperature causes the enzyme to deactivate quickly, making long-term use difficult. Second, since the temperature is higher than room temperature, bubbles are likely to form in the liquid, and the generated air bubbles may adhere to the inner surface of the 70-cell of the detector or the surface of the immobilized enzyme membrane, causing errors in analytical values. . Third, enzyme activity is high at high temperatures, so when using redox enzymes such as glucose oxidase, the amount consumed by the enzyme increases in proportion to the increase in substrate concentration, but when the substrate concentration exceeds a certain level, dissolved oxygen Consuming everything,
The calibration curve deviates from the straight line, and this tendency occurs at lower substrate concentrations as the temperature gets higher. Biological samples and food samples have high substrate glucose concentrations, so they need to be diluted about 100 times, but when the measured concentration is high, [Problems to be solved by the invention] The present invention improves the above problems, extends the life of the enzyme, and widens the measurable concentration range of the substrate. The purpose of this invention is to provide a method that prevents the generation of bubbles in the detector and allows for more accurate analysis than conventional methods.

[Means for solving problems]

In order to achieve this object, the present invention provides an analytical method for measuring a substrate concentration by a reaction rate detection method using an enzymatic reaction, in which a constant temperature bath in which a detector is placed is kept at a predetermined temperature of 250℃ or lower than room temperature. The temperature of the diluted liquid is controlled to the same temperature and supplied to the detector through an air-permeable tube placed in the constant temperature bath.

Analyzers that use enzyme reactions to analyze substrate concentrations using reaction rate detection methods include those that use immobilized enzymes, those that use enzymes in solution, and those that use enzymes in solution. For pz, when using a column filled with a carrier on which an enzyme is immobilized, when using a column packed with a carrier on which an enzyme is immobilized, when using a nylon tube with an enzyme immobilized on an enzymatically active inner wall, and when using a column filled with a carrier on which an enzyme is immobilized, )! 1. When immobilizing the enzyme on a polymer membrane by a physical method, there is a force (there is a force). Also, as a detector, an electrochemical detector, an optical detector, etc. are used. It is also used when using enzymes in solution form!/). The present invention can be applied to any of the above cases. C as air permeable tube
Alternatively, one made of polytetrafluoroethylene or silicone rubber can be used.

[Effect]

In the method of the present invention, the temperature of the constant temperature bath is set to 25r or 25C or lower, which is below room temperature, for analysis. This is to enable enzyme activity to be maintained for a long period of time, and to enable analysis of substrate concentrations up to higher concentrations than conventional ones, as shown in the following examples, and to broaden the analytical range. If analysis is performed at a temperature lower than room temperature (25C or less), the temperature of the diluted solution stored at room temperature will drop, causing the bubbles present in the solution to disappear and forming bubbles on the surface of the A detector, flow cell, immobilized enzyme, etc. This is to prevent this from occurring and to enable highly accurate analysis.

The saturated dissolved oxygen amount of the solution increases as the temperature decreases. When the diluted solution is cooled below room temperature, the amount of dissolved oxygen in the solution can be increased, so when the diluted solution is sent to the detector through an air-permeable tube placed in a constant temperature bath, the dissolved oxygen in the diluted solution is By saturating the oxygen, the oxygen necessary for the redox reaction is supplied, making it possible to analyze highly concentrated substrates and expand the analytical range.

〔Example〕

A phosphate aqueous solution with a pH of 5.6 placed in a container 9 shown in FIG. The diluted liquid was passed through a temperature control coil 6 made by the company (inner diameter 2 mm, outer diameter 3 mm, length 7 m) to maintain the temperature of the diluted liquid at a constant level and to saturate the dissolved oxygen in the diluted liquid. In the thermostat 5, there are a temperature control coil 6, a sample injector, a mixing coil 6', a flow cell containing a stirrer 3, and a flow cell 20.
The attached enzyme electrode 1 is stored in the temperature control coil 6.
The diluted solution passed through the β-
Add 8 μb of D-glucose solution, introduce a certain amount into the flow cell 2 through the mixing coil 6', and stirrer 4. The hydrogen peroxide concentration generated after a certain period of time after being introduced into the flow cell 2 is peaked by the enzyme electrode 1 of the glufus oxidase 5Q mU with the glufus oxidase immobilized membrane attached. It was taken out as a potential and detected as a current value with an amplifier 12 equipped with a differentiation circuit. This result was input into the microcomputer 1a connected to the amplifier 12, and the glucose concentration was calculated. After measurement, the liquid in 70-cell 2 is pumped to waste liquid tank 1 by pump 10.
1, and the substrate concentration was varied. FIG. 2 shows the relationship between the peak potential and glucose concentration at this time. The dilution factor is approximately 500 times.

In FIG. 2, the curve of 25 t::, 10 C assumes that the room temperature is 28 C, 20 tZ' and the constant temperature bath is 250. loC
The 35C curve is obtained when the room temperature is 25C and the constant temperature oven is 35C. 0 According to this result, the straight line part is elongated at low temperature, and 2
It can be seen that in the 5C curve, it is possible to accurately measure substrates that are about 3% thicker than in the case of 35tr, and at 110C, about 11% thicker than in the case of 35t11'.

〔Effect of the invention〕

According to the analytical method of the present invention, it is possible to analyze substrates with higher concentrations than before, expand the analytical concentration range, and prevent the generation of bubbles, making analytical values more accurate. The lifespan can be extended as the temperature of the thermostatic chamber is lower than before.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of an analyzer used in an example of the method of the present invention, and FIG. 2 is a diagram showing the results of the example as a curve of the relationship between peak potential and glucose concentration. Figure 1 Glucose concentration in child's 2nd area (%) Procedural amendment (voluntary) 1. Indication of the case 1982 Patent Application No. 169141 2, Title of the invention Analytical method using enzymatic reaction 3, Amendment Applicant 4, Agent 6 Number of inventions increased by amendment 7, subject of amendment (1) “Enzyme consumption” on page 2, line 18 of the specification is corrected to “oxygen consumption” (2) "Hydrogen peroxide concentration" on page 6, lines 13-14.
Delete "Concentration". (3) "6 Mixing Coil" in Figure 1 of the drawing has been corrected to "6' Mixing Coil" as shown in the attached sheet.

Claims (1)

[Claims] (1) In an analytical method that uses an enzymatic reaction to measure the substrate concentration using a reaction rate detection method, the thermostat in which the detector is placed is kept at a predetermined temperature of 25°C or lower than room temperature, and the diluted solution is An analysis method using an enzyme reaction characterized by supplying the temperature to the detector through an air-permeable tube placed in the constant temperature bath.