JPH0344653B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to chemical species present in an aqueous solution, in particular,
The present invention relates to a method for rapidly, simply, and accurately measuring membrane permeability and diffusion constant for specific chemical species among organic compounds such as sugars, alcohols, and carboxylic acids. The industrial fields of application of the present invention include the polymer chemical industry that manufactures membranes such as separation membranes, the fermentation industry that uses membrane separation processes, the food industry, and the organic chemical industry. It is suitable for the medical and welfare fields in which it is used.

BACKGROUND OF THE INVENTION Membrane permeability and diffusion constants are typically measured through determining the time rate of change in concentration of a chemical species that has passed through the membrane. Conventional techniques for this purpose include methods for estimating concentration changes using changes in absorbance, refractive index, conductivity, etc. ” (November 25, 1982), Baifukan, p210). However, these methods are not highly selective for specific chemical species, and in practice, it is difficult to detect membrane permeation of specific chemical species, especially when a large number of components coexist. It has the disadvantage of being impossible.

Problems to be Solved by the Invention The present inventors have developed a method for quickly, simply, and accurately measuring the permeability of membranes to specific chemical species in aqueous solutions containing many components such as fermentation liquids and body fluids. In order to develop a method to obtain hydrogen peroxide, we have conducted extensive research and found that, using the above chemical species as one of the substrates, we immobilized an oxidase that produces a small amount of co-hydrogen peroxide as one of the products. By using an enzyme electrode consisting of a membrane and an enzyme electrode or a hydrogen peroxide electrode, we can determine the concentration of chemical species that have passed through the membrane by determining the rate of change over time in the response current output of this enzyme electrode. found that a method for estimating the rate of change over time can be suitable for that purpose,
Based on this knowledge, the present invention was accomplished.

Means for Solving the Problems That is, the present invention provides a method for measuring the permeability and diffusion constant of a membrane for chemical species present in an aqueous solution, in which one of the two aqueous solution phases separated by the membrane is used. A membrane having the above chemical species as one of the substrates and an oxidizing enzyme that produces a small amount of co-hydrogen peroxide as one of the products from this and oxygen is immobilized on the phase, and an oxygen electrode or a hydrogen peroxide electrode. by inserting an enzyme electrode consisting of a combination of The present invention provides a method for measuring the transmittance and diffusion constant of a membrane, characterized by determining the transmittance and diffusion constant of the membrane.

The oxidases used in the method of the present invention are code numbered EC1, X, 3, Y (X is a natural number from 1 to 100, Y is any natural number ), or enzymes similar thereto, which use the target chemical species as a substrate can be arbitrarily selected.
For example, if the target chemical species is glucose, glucose oxidase (EC1.1.3.4),
Catechol oxidase (EC1.1.3.14) if it is catechol, pyruvate oxidase (EC1.2.3.3 or EC1.2.3.3 or
EC1.2.3.6) can be used conveniently.
Furthermore, when there is no oxidase that uses the target chemical species as one of its substrates, other enzymes may be used as an auxiliary together with the oxidase. For example, if the target chemical species is sutucarose, invertase (EC3.2.1.26), mutarotase (EC5.1.3.3)
is advantageously used in combination with glucose oxidase.

The enzyme immobilization method used in the inventor's method may be any of the commonly used covalent bonding methods, ionic bonding methods, entrapment methods, adsorption methods, etc., and the above oxidizing enzyme may be immobilized by any of these methods. It is immobilized on a membrane and attached to an oxygen electrode or hydrogen peroxide electrode to form an enzyme electrode. The oxygen electrode used in the method of the present invention includes metal electrodes such as platinum, gold, silver, and nickel;
Alternatively, commonly used Clark-type electrodes can be used. As the hydrogen peroxide electrode, a noble metal electrode such as platinum or gold, a metal oxide electrode such as tin oxide or indium oxide, or a commonly used electrode whose surface is covered with a porous polymer film can be used.

In the method of the present invention, the measurement cell is usually configured such that the membrane sample whose transmittance and diffusion constant are to be measured is sandwiched between two tanks. There is no particular restriction as long as the electrode can be inserted. However, the aqueous solution placed in the measurement cell is a near-neutral buffer solution that is kept at a temperature of about 20 to 37°C so that the enzyme electrode can fully perform its function and the enzyme used will not be deactivated. It is preferable.

The principle of the method for measuring membrane transmittance and diffusion constant of the present invention is as follows. The chemical species diffuses through the membrane from one aqueous solution phase to which the chemical species have been added to the other aqueous solution phase in which the enzyme electrode is inserted. The increase in the concentration of the chemical species toward the enzyme electrode insertion side due to this diffusion is immediately detected as a change in the response current at the enzyme electrode. Therefore, the rate of increase in the concentration of the chemical species due to diffusion is determined from the rate of change over time in the response current, and the permeability and diffusion constant of the membrane for the chemical species are measured.

Next, the method of the present invention will be explained in more detail with reference to the accompanying drawings.

FIG. 1 is a schematic cross-sectional view showing an example of an apparatus used in the measuring method of the present invention. There are two aqueous phases 2 and 3 across the membrane 1 whose permeability and diffusion constant for a chemical species are to be measured. The aqueous solution is stirred using a magnetic stirrer 10, for example. In one aqueous solution phase 3, an enzyme electrode is inserted, in which a membrane 5 is attached to the surface of an enzyme electrode or a hydrogen peroxide electrode 4, on which an oxidase having the above chemical species as one of its substrates is immobilized. The output current from the enzyme electrode is measured by an ammeter 6 and recorded by a recorder 7. Enzyme electrode or peroxide electrode 4
If a Clark-type battery-type enzyme electrode is not used, a reference electrode 11 such as a silver-silver chloride electrode is used.
A counter electrode 12 made of a platinum wire or the like is also inserted into the aqueous solution phase 3, and an appropriate potential is applied to the enzyme electrode or hydrogen peroxide electrode 4 using a constant potential power source 8.
It is necessary to ensure sufficient reduction of oxygen or oxidation of hydrogen peroxide. Regarding the potential value, when using a silver-silver chloride electrode as a reference electrode, for oxygen reduction, -
Appropriate values are around 0.8V, and for hydrogen peroxide oxidation, around +0.6V with respect to the reference electrode.
Before adding the sample containing the above chemical species to the aqueous solution phase 2, the concentration of this chemical species in the aqueous solution phase 3 is zero, so when an oxygen electrode is used as the electrode 4, the concentration of this chemical species from the aqueous solution phase 3 It shows a constant current value that is proportional to the diffusion rate of oxygen to the electrode surface, that is, the dissolved enzyme concentration in the aqueous solution phase 3. When a hydrogen peroxide electrode is used, the current value is zero because there is no hydrogen peroxide in the aqueous solution phase. shows a current value close to .

When a predetermined amount of the sample solution containing the above-mentioned chemical species is added to the aqueous solution phase 2 of the device prepared in this manner using, for example, a microsyringe 9, the added chemical species permeate through the membrane 1 and enter the aqueous solution phase 3. reach.
In the aqueous phase 3, the chemical species are present on the immobilized enzyme membrane 5.
As a result, the oxygen concentration near the electrode 4 decreases and the hydrogen peroxide concentration increases. Accordingly, when an oxygen electrode is used as the electrode 4, the output current decreases, and when a hydrogen peroxide electrode is used, the output current increases.

Here, if the concentrations of the chemical species in the aqueous solution phases 2 and 3 are C _A and C _B , respectively, and the cross-sectional area and thickness of the film 1 are S and d, then the chemical species in the film 1 are
The relationship between the amount J passing through per unit time Δt, the permeability P and the diffusion constant D of the membrane 1 is expressed by the following equation (for example, "Membranes and Ions" by Tetsuya Hanai (Showa)
53.11), Kagaku Doujin, p.40).

JSΔt=P(C _A −C _B )SΔt=(D/d) (C _A −C _B ) …(1) Here, C _A is a known concentration, C _B is zero, or a known concentration, and S and d is a measurable quantity, so
If J is known, P and D can be found. J
can be determined, for example, by the following procedure. The ratio of the output current change (Δi) to the substrate concentration C (Δi/ Calculate C) separately and calibrate the enzyme electrode. Thereafter, in the apparatus _shown in _{FIG. 1, the rate of temporal change in the output current of the enzyme electrode (Δi 2 /} Find Δt). If the volume of the aqueous solution phase 3 is V, then J is expressed by the following formula.

J=(Δi ₁ /C) ⁻¹ (Δi ₂ /Δt)·S ⁻¹ V (2) Therefore, P and D are given by the following equations.

P=Δi ₁ ^-1 i ₂ C(C _A −C _B ) ⁻¹ Δt ⁻¹ Δt ⁻¹ S ⁻¹ V …(3) D=Δi ₁ ⁻¹ Δi ₂ C(C _A −C _B ) ⁻¹ Δt ^-1 S ^-1 Vd...(4) In this way, the permeability and diffusion constant of the membrane for chemical species can be measured.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Measurement of transmittance and diffusion constant of photocrosslinkable polyvinyl alcohol membrane for lactic acid Lactate oxidase (Boehringer Mannheim, EC1.13.12.4, 15U/mg at pH 7.0
It was dissolved in 0.3 ml of 0.02M phosphate buffer solution and photocrosslinkable polyvinyl alcohol (Kunihiro Ichimura, "Journal of Polymer Science, Polymer Chemistry Edition" (J.Polym.Sci.)
Polym.Chem Ed) Volume 22. P2817−2828, 1984)
The mixture was mixed with 0.7 ml, spread on an area of about 6 cm ² , air-dried, and further irradiated with 20 mW/cm ² xenon arc lamp light for 10 minutes to obtain a water-insoluble lactate oxidase-immobilized membrane. The diameter of the immobilized membrane is approximately 1
This was cut out to a size of 0.06 cm ² and placed on the diaphragm of a Clark-type battery-type oxygen electrode (Ishikawa Seisakusho DG-1 model), which has a platinum electrode with an area of about 0.06 cm 2 as the cathode and a porous polytetrafluoroethylene membrane as the diaphragm. It was tightened and fixed with an O-ring to obtain a lactate oxidase-immobilized enzyme electrode. Separately, the same photocrosslinkable polyvinyl alcohol alone was developed, air-dried, and irradiated with light to obtain a sample film. The thickness of the sample membrane is 0.008 cm in the swollen state. Two aqueous solution phases separated by a sample membrane with an effective surface area of 1 cm ² each contained 10 ml of a 0.1 M phosphate buffer solution, and the enzyme electrode obtained as described above was inserted into one of the aqueous solution phases. Both phase solutions were stirred using a magnetic stirrer and the temperature was maintained at 25°C.

Figure 2 shows the output at the oxygen electrode when 0.05mM lactic acid was added to the aqueous solution phase on the side where the enzyme electrode was inserted in the measurement system, and 2 minutes later, 0.2M lactic acid was added to the aqueous solution phase on the other side. It is a graph showing a change in current over time. After adding lactic acid to the aqueous solution phase on the side where the enzyme electrode is first inserted, the enzyme electrode is calibrated based on the relationship between the steady current value subtracted from the initial current value after about 1 minute and the lactic acid concentration added at this time. can do. Next, if we calculate the time rate of output current decrease in the part where the output current of the enzyme electrode decreases linearly with time after adding lactic acid to the other aqueous solution phase, we can obtain equations (3) and (4). Therefore, the permeability and diffusion constant of the sample membrane for lactic acid are 3.1×
Values of 10 ⁻⁵ cms ⁻¹ and 2.5×10 ⁻⁷ cm ² s ⁻¹ can be obtained. Using the above measurement system, we used three types of lactic acid beverages as lactic acid-containing solutions, and determined the transmittance and diffusion constant of the sample membrane for lactic acid, which were 2.8 to 3.1×10 ^-5 cms ^-1 and 2.2, respectively. ~2.5×10 ^-7 cm ² s ^-1
obtained the value of

Example 2 Measurement of permeability and diffusion constant of triacetylcellulose-containing membranes for glucose.

10 mg of glucose oxidase (Boehringer Mannheim, EC1.1.3.4, 250 U/mg) was immobilized in a photocrosslinkable polyvinyl alcohol film in the same manner as in Example 1. The diameter of the immobilized membrane is approximately 1.5
This was cut out to a size of 1 cm, placed on the surface of a platinum electrode with an area of about 0.2 cm ² , and fixed by tightening with an O-ring to obtain a glucose oxidase-immobilized enzyme electrode. on the other hand,
The sample membrane was prepared using the method described in the literature (Masao Koyama et al.
"Anaretica Simica Acta" Volume 116, P307,
(1980), triacetylulose, 1.8−
A membrane consisting of diamino-4-aminomethyloctane and glutaraldehyde was prepared, and this membrane was further subjected to reduction treatment in an aqueous sodium borohydride solution to obtain a sample membrane. The thickness of the sample membrane is 0.0033 cm in the swollen state. Two aqueous solution phases are formed through a sample membrane with an effective surface area of 1 cm ² , each consisting of 10 ml of 0.1 M phosphate buffer solution. Dempa Kogyo HS-907 model) and a platinum wire electrode, connect these electrodes to the working electrode, reference electrode, and counter electrode connectors of a constant potential power supply (Hokuto Denko HA-501 model), and refer to the potential of the working electrode. It was regulated to +0.65V with respect to the pole. The two phase solutions are stirred together using a magnetic stirrer;
Its temperature was kept at 25℃.

Figure 3 shows the output current at the enzyme electrode when 0.1mM glucose was added to the aqueous solution phase on the side where the enzyme electrode of the measurement system was inserted, and 2 minutes later, 50mM glucose was added to the aqueous solution phase on the other side. It is a graph showing the temporal change of. In this example, unlike Example 1, hydrogen peroxide is detected with an electrode, so the output current of the enzyme electrode increases with the addition of glucose. The permeability and diffusion constant of the sample membrane for glucose are 1.9 × 10 ^-5 cm, respectively.
A value of s ^-1 and 6.3×10 ^-7 cms ^-1 is obtained.

Figure 4 shows the concentration by adding glucose appropriately.
This is a graph showing the results of repeated measurements of the diffusion constant of the sample membrane for glucose using the method described above, using a 1/4 diluted serum of 20 mM in the aqueous phase on the side where the enzyme electrode is not inserted. It can be seen that as the number of measurements increases, the diffusion constant decreases slightly due to protein adsorption to the sample membrane surface.

[Brief explanation of drawings]

FIG. 1 is a schematic cross-sectional view showing an example of an apparatus for measuring transmittance and diffusion constant of a membrane according to the present invention, and FIG.
FIG. 3 is a graph showing temporal changes in output current in a lactate oxidase-immobilized enzyme electrode for the purpose of determining the permeability and diffusion constant of a sample membrane for lactic acid by the method of the present invention. FIG. 4 is a graph showing temporal changes in output current at a glucose oxidase-immobilized enzyme electrode for the purpose of determining glucose permeability and diffusion constant of a sample membrane using the method.
1 is a graph showing the results of repeated measurements of the diffusion constant of a sample membrane for glucose in serum using the present method of the present invention. In Figure 1, 1 is the sample membrane, 2 and 3 are the aqueous solution phases separated by the sample membrane, 4 is the enzyme electrode or hydrogen peroxide electrode, 5 is the oxidase-immobilized membrane, 6 is the ammeter, and 7 is the recorder. 8 is a constant potential power supply, 9 is a microsyringe for supplying chemical species, 10 is a magnetic stirrer, 11 is a reference electrode, and 12 is a counter electrode.

Claims (1)

[Claims] 1. In a method for measuring the permeability and diffusion constant of a membrane for chemical species present in an aqueous solution, one of the two aqueous solution phases partitioned by the membrane is provided with a membrane for the chemical species. By inserting a responsive enzyme electrode, adding a sample containing the above chemical species to the other phase, and then determining the rate of change over time in the response output of the enzyme electrode, the permeation of the membrane can be determined. A method for measuring the transmittance and diffusion constant of a membrane, the method comprising measuring the permeability and diffusion constant of a membrane. 2. The enzyme electrode comprises a membrane immobilized with an oxidizing enzyme that uses the chemical species and oxygen as substrates and gives at least hydrogen perfermented product as one of the products, and an oxygen electrode or a hydrogen peroxide electrode. A method for measuring the transmittance and diffusion constant of a membrane according to claim 1, which comprises: