JPH0697221B2 - Method for measuring organic concentration and apparatus used for the method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a rapid quantification method of an organic substance using an enzyme electrode equipped with an immobilized membrane of an enzyme that specifically reacts with an organic substance such as alcohol, and The present invention relates to a device used in a method.

[Conventional technology]

Conventionally, as a method and apparatus for measuring the concentration of organic substances such as alcohol with an enzyme electrode, for example, a sample is diluted as a pretreatment for measuring the alcohol concentration of an alcoholic beverage, and then a constant current value for a constant concentration of alcohol, for example. (I.e., the steady-state rate of the enzymatic reaction), and the method of calculating the alcohol concentration from the rate or acceleration until the steady-state current value after dilution of the sample is proposed or used (Am. J.Eno
l.Vitic.Vol34, No.3 (1983), 173-175, Analytica C
himica Acta75 (1975) 169-180, Japan Brewing Association Magazine 80
Volume 3 (1985) 206-207, Appl. Microbiol Biotechn
ol 19 (1984) 181-185, Biotechnology and Bioeng.
ineering Vol25 (1983) 1049-1055, Annals New Yo
rk Academy of Sience Vol.434 (1984) 515−51
9).

Furthermore, there is a flow injection method in which an enzyme electrode is installed in a flow cell, a constant flow rate of a buffer solution is always flown, a sample is diluted, and a small amount of sample is injected into a line to measure a steady-state current value (Analyst , Vol, 106 (1981) 1309
-1317), and a flow injection device (glucose analyzer manufactured by Toago Denki Co., Ltd.) that does not require dilution.

[Problems to be solved by the invention]

In the conventional method of measuring the steady-state current value for organic matter,
The quantifiable range is narrow, within a few hundred ppm, and it is necessary to dilute the sample for normal organic concentration measurement. Further, even the above-mentioned flow injection method which does not require dilution has a problem that a precise cell having a small volume and a micro stirrer for stirring the liquid in the cell are required.

The present invention is based on the above background,
It is an object of the present invention to provide a measurement method which does not require pretreatment such as sample dilution, has a wide measurable concentration range, and allows rapid measurement, and an apparatus used for the method. .

[Means for solving problems]

As a result of conducting various tests and studies on a quantitative analysis method of organic concentration, the inventors of the present invention have constantly flown a buffer solution at a constant flow rate into the enzyme electrode, injecting a small amount of sample into the line, and It is effective to achieve the object of the invention if not the total amount but a part of it is reacted with the enzyme electrode and the total amount of organic substances involved in the enzymatic reaction is measured by the obtained integrated value of the response current to obtain the organic concentration in the sample. Is found,
The present invention has been completed.

The apparatus for measuring the concentration of an organic substance such as alcohol of the present invention comprises: (a) a flow cell section having an inlet for a buffer solution flow, an enzyme electrode and an outlet for the buffer solution flow, and (b) a buffer solution at a constant flow rate. A liquid feeding device that feeds a liquid to the inlet of the flow cell via a conduit, and (c) a sample injection unit that is provided in a conduit between the flow cell and the liquid feeding device and that injects a certain amount of an organic-containing sample. ) A temperature control device that adjusts the liquid temperature in the conduit between the flow cell and the liquid feeding device and in the flow cell to a constant temperature, and (e) reacts the organic matter in the injected sample with the enzyme electrode and A measuring unit for detecting and measuring the generated response current, and an organic concentration measuring device comprising: (a) a cylindrical vertical flow cell having a buffer solution inlet at the bottom; Vertical cylinder type -The enzyme electrode fitted in the cell (however, the outer diameter of this enzyme electrode is sufficiently larger than the inner diameter of the cylindrical vertical flow cell, and the liquid flow flowing in from the inflow port is a thin layer). It has a space between it and the inner wall of the flow cell so that it flows along the electrode, and this electrode has an immobilized enzyme membrane surface at its lower end, and this immobilized enzyme membrane surface enters from the inflow port. It is arranged so that the incoming liquid flow strikes vertically and faces the inflow port closely.), (C) The inflow port is defined by the inner wall of the flow cell and the outer wall of the enzyme electrode. A drainage chamber in which the liquid flow is made to flow after coming into contact with the immobilized enzyme membrane, and (d) a drainage port provided above the drainage chamber. A portion of the organic matter in the sample Together so as to knowledge, a device characterized by a structure so as to measure the integrated value by the measurement unit.

The present invention will be described in more detail.

Organic Concentration Measuring Method The object to be measured in the present invention is an organic substance-containing sample, and examples of such organic substances include alcohol, glucose, galactose, lactic acid and the like. In addition, as an organic matter-containing sample that can be used in the present invention,
There are regular beer, other special beer, various alcoholic beverages such as sake, and beverages containing sugar and lactic acid.

The enzyme electrode used in the present invention is one in which an enzyme that specifically and selectively reacts with an organic substance to be measured is immobilized. For example, when the organic substance is alcohol, the enzyme alcohol oxidase is added to a DEAE cellulose membrane. It was fixed on a platinum electrode, mounted on a platinum electrode, and covered with a polycarbonate film to prepare a detection part for the enzyme electrode.
In this example, the concentration of hydrogen peroxide, which is the product of the enzymatic reaction, can be converted into a current value. The present invention is not limited to the example relating to this alcohol, for example, when the subject is glucose, glucose oxidase is used as an enzyme, and when the subject is galactose, galactose oxidase is used as an enzyme. It can be applied to the measurement of organic matter.

One of the characteristics of the measuring method of the present invention is that only a predetermined partial amount of the organic matter in the sample injected into the buffer flow having a constant flow rate reacts with the enzyme electrode, and the response current generated from the enzyme electrode by the reaction is The integrated value is measured, and the organic concentration is calculated based on the integrated value. Next, a measuring device that can exhibit such characteristics will be described.

Organic Concentration Measuring Device First, the measuring device of the present invention is schematically illustrated as an example.
It will be described with reference to the drawings.

This device 1 supplies a cylindrical vertical flow cell 2, an enzyme electrode 3 inserted in the flow cell 2, and a buffer solution from a buffer solution tank 4 to a bottom inlet port 6 of the enzyme electrode 3 via a conduit 5. A liquid sending device (pump) 7 for liquefying, a sample injecting section 8 provided in the conduit 5 for injecting a fixed amount of the organic-containing sample into the buffer solution flow, and the temperature of the liquid in the conduit 5 and the flow cell 2 are adjusted. And a measuring unit 10 for detecting a response current generated from the enzyme electrode 3 due to an enzyme reaction and measuring an integrated value thereof. FIG. 2 shows a partially enlarged sectional view of the flow cell and the enzyme electrode of this device example. As shown in FIG. 2, this flow cell has an inlet 6 for a buffer solution flow 11 at the bottom and a drainage port 12 at the top, and this enzyme electrode 3 has an immobilized enzyme membrane at the lower end. It is provided with 13 and is inserted into the inside of the flow cell 2 so that the lower surface of the immobilized enzyme membrane 13 is arranged above the inflow port 6. Further, it has a drainage chamber 14 defined by the inner wall of the flow cell 2 and the outer wall of the enzyme electrode 3, and a drainage port 12 is provided above the drainage chamber.
Is provided.

The flow cell of the device of the present invention is of vertical type, preferably of small capacity. Although it depends on the flow rate of the buffer solution, the sample injection rate, etc., for example, when the buffer solution flow rate is 5 ml / min and the sample injection rate is 5 μ, the outer diameter of the enzyme electrode is 14.4 mmφ and the inlet diameter is 1 mm.
With φ, the inner diameter of the flow cell can be 14.6 to 15.4 mmφ and the distance from the inlet to the electrode can be 1 to 4 mm.

[Work]

In the present invention, a buffer flow having a constant flow rate is applied to the surface of the immobilized enzyme membrane to supply the enzyme electrode with the solution, and the system is immediately drained,
When an organic-containing sample is injected into the buffer solution flow before this liquid supply,
The organic matter mixes uniformly with the buffer solution and surely and uniformly hits the surface of the immobilized enzyme membrane together with the buffer solution. There is an enzyme that reacts specifically and selectively with the organic matter on the surface of the immobilized enzyme membrane. When the organic substance reacts with the enzyme on that side and the resulting product is, for example, an oxidizing substance, it is detected as a response current at the redox electrode. In this invention, since the buffer solution flow is supplied to the enzyme electrode and immediately drained,
Only a certain proportion of the injected organic matter reacts with the enzyme, not the total amount. The drainage flows into the drainage chamber as it is through the gap between the cell and the enzyme electrode, and is discharged from the drainage port as a stable flow. The detected response current corresponds to the reaction rate of the enzyme reaction, and the concentration was conventionally measured from the peak value of this current value, but from the integrated value of the response current obtained in the present invention, the organic matter involved in the enzyme reaction was measured. Then, the organic concentration in the injected sample is calculated. This is because the integrated value has a linear relationship with the concentration of organic matter in the sample.

The operation and action of the present invention will be described with the device of the present invention.

The buffer solution is supplied to the inflow port 6 of the flow cell 2 through the conduit at a constant flow rate by the liquid supply device (pump 7) and is made to flow into the flow cell. The buffer solution surely and uniformly hits the immobilized enzyme membrane surface of the enzyme electrode above it from the inflow port 6 below it, and is immediately drained. The effluent flows into the effluent material 14 through the gap between the cell and the enzyme electrode as it is, and is ejected from the effluent port to form a stable flow. A fixed amount of organic material-containing sample is injected into the buffer solution flow from a sample injection part 8 provided in a conduit between the flow cell and the liquid delivery device, and the organic material is mixed with the buffer solution and immobilized enzyme membrane surface 13
As described in the above method, only a part of the injected organic matter reacts with the enzyme, and the organic matter concentration in the injected sample is calculated.

〔The invention's effect〕

The following effects can be obtained by the concentration measuring method and apparatus of the present invention.

(A) Since a small amount of sample is directly injected into the buffer flow,
For example, the alcohol concentration range that can be quantified broadly extends to 0 to 25%, and the sample dilution operation is not required.
It can be used as a so-called online type organic sensor.

(B) Since the integrated value of the response current has a good linear relationship with the concentration of the organic substance in the sample, the organic substance can be measured accurately and quickly.

〔Example〕

 The present invention will be specifically described by the following examples.

Example 1 Immobilization in which a DEAE cellulose membrane (Whatman thickness 120 μm) was impregnated with an alcohol oxidase solution and washed with a phosphate buffer solution was covered with a polycarbonate membrane (Nuclepore membrane manufactured by Nomura Microscience, pore size 0.03 μm, thickness 5 μm). Enzyme membrane and H ₂ O ₂ electrode (Toa Denpa Kogyo, PS
-115C) was assembled as shown in FIG. 2 and the device was assembled as shown in FIG.

0.1M phosphate buffer solution was flowed from the buffer tank to the flow cell at a flow rate of 5.2 ml / min through a conduit, and a standard ethanol solution of known concentration was injected from the sample injection part (injector) prior to sample measurement. Calculate the integrated current value. From this standard value, the integrated current value of the sample thereafter is converted by proportional calculation to calculate the alcohol concentration of the sample.

Regular beer (Kirin beer, light beer) was used as a sample, and the alcohol concentration of the beer was measured under the following measurement conditions. In addition, the alcohol concentration of the sample was measured in parallel by the distillation method which has been established as an accurate method for measuring alcohol concentration. The results are shown in Table 1 with the value obtained by the method according to the present invention as the measured value and the value obtained by the distillation method as the true value.

Measurement conditions Flow velocity 5.2 m / min Temperature 20.0 ° C. pH 8.00 (0.1 M phosphate buffer) Injection amount 5 μ Examples 2 to 11 Example 1 was similarly measured except that the samples shown in Table 1 were used. The results are shown in Table 1.

From the above results, the measurement error was ± 0.1% in alcohol concentration in all cases.

The response time was within 5 to 6 minutes.

[Brief description of drawings]

FIG. 1 is an explanatory view showing the outline of an example of a measuring apparatus that can be used in the method of the present invention, and FIG. 2 is a partial sectional view showing the flow cell and the enzyme electrode of FIG. 1 ... Measuring device, 2 ... Flow cell, 3 ... Enzyme electrode,
4 ... buffer tank, 5 ... conduit, 6 ... inlet, 7 ...
… Pump, 8 …… Sample injection part (injector), 9…
… Constant temperature bath, 10 …… Measuring part, 11 …… Buffer flow, 12 …… Drainage port, 13 …… Immobilized enzyme membrane, 14 …… Drainage chamber

Claims (2)

[Claims] 1. A flow cell section having (a) a buffer solution inlet, an enzyme electrode and a buffer solution outlet, and (b) a delivery means for delivering the buffer solution at a constant flow rate to the inlet of the flow cell via a conduit. A liquid device, (c) a sample injection section provided in a conduit between the flow cell and the liquid sending device, for injecting a fixed amount of the organic-containing sample, and (d) in a conduit between the flow cell and the liquid sending device, and A temperature control device for adjusting the liquid temperature in the flow cell to a constant temperature, and (e) a measuring unit for reacting organic substances in the injected sample with an enzyme electrode to detect and measure a response current generated from the enzyme electrode. An organic-concentration measuring apparatus comprising the following: the flow cell section and the measuring section are constituted by the following members. (A) A cylindrical vertical flow cell having an inlet for a test solution composed of a sample-containing buffer solution at the bottom, (b) an enzyme electrode fitted in the cylindrical vertical flow cell (however, the enzyme electrode is the outside thereof). The diameter is sufficiently larger than the inner diameter of the cylindrical vertical flow cell, and there is a gap between the inner wall of the flow cell so that the liquid flow flowing in from the inlet flows as a thin layer along the electrode. In addition, this electrode has an immobilized enzyme membrane surface at its lower end, so that the immobilized enzyme membrane surface hits the liquid stream flowing in from the inlet vertically and in the vicinity of the inlet. (C) is defined by the inner wall of the flow cell and the outer wall of the enzyme electrode, and the liquid flow after flowing through the inflow port and coming into contact with the immobilized enzyme membrane is A drainage chamber that is distributed, (d) A drainage port provided above the liquid chamber, (e) The response current generated by reacting a part of the organic matter in the test liquid flow with the enzyme electrode is detected, and the integrated value thereof is measured. Measuring section. 2. A method for measuring an organic concentration in a sample by using the following organic concentration measuring device, wherein a test liquid containing the sample is flown from the test liquid inlet of the flow cell at a sufficiently fast rate and fixed. A method for measuring an organic matter concentration, which comprises immediately applying the deoxidized enzyme membrane surface to a drainage chamber, and then immediately discharging it. Organic concentration measuring device (a) A flow cell part having a buffer solution inlet, an enzyme electrode and a buffer solution outlet, and (b) a liquid transfer solution that delivers the buffer solution at a constant flow rate to the flow cell inlet via a conduit. Device, (c) a sample injection part for injecting a fixed amount of organic-containing sample provided in a conduit between the flow cell and the liquid feeding device, and (d) in the conduit between the flow cell and the liquid feeding device and the flow cell A temperature control device for adjusting the liquid temperature in the inside to a constant temperature, and (e) a measuring unit for reacting organic substances in the injected sample with an enzyme electrode to detect and measure a response current generated from the enzyme electrode, An organic-concentration measuring device comprising the flow cell part and the measuring part made of the following members. (A) A cylindrical vertical flow cell having an inlet for a test solution composed of a sample-containing buffer solution at the bottom, (b) an enzyme electrode fitted in the cylindrical vertical flow cell (however, the enzyme electrode is the outside thereof). The diameter is sufficiently larger than the inner diameter of the cylindrical vertical flow cell, and there is a gap between the inner wall of the flow cell so that the liquid flow flowing in from the inlet flows as a thin layer along the electrode. In addition, this electrode has an immobilized enzyme membrane surface at its lower end, so that the immobilized enzyme membrane surface hits the liquid stream flowing in from the inlet vertically and in the vicinity of the inlet. (C) is defined by the inner wall of the flow cell and the outer wall of the enzyme electrode, and the liquid flow after flowing through the inflow port and coming into contact with the immobilized enzyme membrane is A drainage chamber that is distributed, (d) A drainage port provided above the liquid chamber, (e) The response current generated by reacting a part of the organic matter in the test liquid flow with the enzyme electrode is detected, and the integrated value thereof is measured. Measuring section.