JPS61281960A - Alcohol sensor of hybrid enzyme film - Google Patents

Abstract

PURPOSE:To effectively block a carboxylic acid even with a thin film having a relatively large pore size and to prevent the response of a sensor by using a thin film of a hydrophobic polymer as a barrier layer. CONSTITUTION:This alcohol sensor is constituted by mounting an alcohol oxidase immobilized film on a physicochemical device and is used to detect the alcohol lin an aq. soln. The molecules of the alcohol to be measured in the soln. are made to permeate through the sensor. The thin film of the hydrophobic polymer having fine pores of <=15mum pore size and >=25% porosity is deposited on the alcohol oxidase immobilized film. The measurement up to the high alcohol concn. and the expansion of the measurement range are made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly selective alcohol sensor, ie, an alcohol sensor that is not affected by coexisting carboxylic acids. More specifically, the present invention relates to an alcohol sensor whose main feature is the construction of a permeable membrane applied to a physicochemical device.

PRIOR ART AND PROBLEMS THEREOF Physicochemical devices 1, for example alcohol sensors consisting of an acid 1A electrode covered with an alcohol oxidase immobilized membrane, are known [Anal, Chill, Acta]; 75 (
1975) 169-180).

When alcohol in the test solution diffuses into the alcohol oxidase-immobilized membrane together with dissolved oxygen and is catalytically oxidized by this enzyme, the consumption of oxygen there is reduced by the oxygen electrode. It is based on the principle of being detected. Alcohol oxidase-immobilized membranes can be made from various types of membranes that are suitable from the viewpoint of function and production, such as alcohol oxidase solution and hydrophilic polymer II! (for example, an acedel cellulose membrane), and others. Wt cord electrodes themselves are also known, for example, the so-called Clark type, that is, a lead electrode as an anode, Others are used, including those in which a platinum electrode is used as the cathode, a highly concentrated alkaline solution is used as the electrolyte, and the platinum electrode is covered with an oxygen gas permeable thin film (for example, a polytetrafluoroethylene film). Oxygen electrode and fermentation using it
For details or specific examples of electrodes, see "Ion Electrodes and Enzyme Electrodes" (Kodansha Scientific).

Please refer to pages 77 et seq.

Since such an alcohol oxidase-immobilized membrane-attached enzyme electrode utilizes the action of an enzyme, substances other than alcohol should not be detected due to the substrate specificity of the enzyme. However, perhaps because the enzyme preparations used are not necessarily highly pure, they tend to digest carboxylic acids in the test solution, and the selectivity of alcohol detection is not satisfactory. is observed.

Possible Solution If the alcohol detection principle of an alcohol sensor using an alcohol oxidase-immobilized membrane is as described above, then in order to make carboxylic acid act as this alcohol oxidase, carboxylic acid is generally larger in size than alcohol. Focusing on this, it is conceivable to attach a polymer i111i having small pores as a barrier layer to the alcohol oxidase immobilized membrane and block the carboxylic acid with the barrier layer.

However, such solutions have not been successful.

That is, the pore diameter is 0.2 μm and the film thickness is 150 μm.
This is because even if a μm thick acedelcellulose membrane was used as the barrier layer, the response to acetic acid when acetic acid coexisted with ethanol was the same as when no barrier layer was used (see experimental example below).

SUMMARY OF THE INVENTION Once the present invention aims to provide a solution to the above-mentioned points, it seeks to achieve this object by using a thin film of a hydrophobic polymer as the above-mentioned barrier layer.

Therefore, the highly selective alcohol sensor according to the present invention
In an alcohol sensor for detecting alcohol in an aqueous solution, which consists of an alcohol oxidase immobilized membrane mounted on a physicochemical device, a pore with a pore diameter of 15 μm or less is used to allow the molecules of the alcohol to be measured in the solution to pass through. It is characterized in that a thin film of a hydrophobic polymer having a porosity of 25% or more is adhered to the alcohol oxidase immobilized membrane.

According to the present invention, even a thin film with a relatively large pore size can effectively block carboxylic acid and prevent sensor response.

That is, when a porous polytetrafluoroethylene membrane with a pore diameter of 6.7 μm and a film thickness of 95 μm was used as a barrier layer, the response to acetic acid was zero when acetic acid was coexisting with ethanol. In this case, the acetyl cellulose membrane described above, which should have a harder diameter and length than the pores through which acetic acid passes through, could not block the response of acetic acid at all, so a hydrophobic polymer membrane was used as a barrier layer. This effect seen when used should be considered unexpected.

In addition, conventional alcohol sensors have a narrow detection range and can only measure alcohol concentrations in the low concentration range.
According to the sensor of the present invention, it is possible to measure alcohol concentrations up to high levels that could not be measured conventionally. It should be said that the fact that such an expansion of the measurement range became possible was unexpected.

Detailed Description of the Invention The alcohol sensor of the present invention is defined as above, and is an alcohol sensor for detecting alcohol (in an aqueous solution) comprising an alcohol oxidase immobilized membrane mounted on a physicochemical device. This is a proprietary method in which an oxidase-immobilized membrane is coated with a specific porous hydrophobic polymer thin film.

Here, [A thin film of a hydrophobic polymer is coated with the alcohol oxidase-immobilized film] means that the thin film of a specific hydrophobic polymer is coated by directly contacting the alcohol oxidase-immobilized film of the basic alcohol sensor. When worn and other porous thin I! This includes any case in which a layer (including a network) that allows the passage of alcohol is interposed and deposited. Such intervening layers include cellulose membranes, cellophane membranes, polyvinyl alcohol membranes, porous polyvinyl chloride membranes, polyamino acid membranes, and others.

Basic Alcohol Centers It has been mentioned above that the alcohol centers to be improved according to the present invention are generally known.

Typical physicochemical devices used as basic alcohol sensors include oxygen electrodes, hydrogen peroxide electrodes, and semiconductor devices. Oxygen electrodes and hydrogen peroxide electrodes are also often used behind these electrodes.

The oxygen electrode having the structure described above is typical.

The al'-ru oxidase-immobilized membrane that should have the function of an al'-ru sensor in this physicochemical device is
A solution of this enzyme, especially an aqueous solution, is poured into a suitable porous thin film material.
In particular, it is impregnated with hydrophilic materials such as acetylcellulose, nitrocellulose, porous polyvinyl chloride, polyvinyl alcohol, polyamino acids, etc., or film-formed and insolubilized with a solution of a polymer (e.g., sodium alginate) in which an enzyme is dissolved. (for example, contacting calcium or aluminum ions with sodium alginate).

Barrier According to the present invention, a specific hydrophobic porous membrane is applied directly or indirectly to the alcohol oxidase immobilized membrane.

The thin film used as the Burr V layer according to the invention is made of a hydrophobic polymer having a porosity of 25% or more and having pores with a diameter of 15 μm or less. The lower limit of the pore diameter is
Needless to say, the number of molecules of alcohol in the solution to be measured must be sufficient to pass through these pores and reach the alcohol oxidase immobilized membrane. It goes without saying that a larger number of pores is preferable as long as the strength of the thin film is not excessively reduced. Therefore, this thin film has a porosity of 25% or more. Here, "porosity" means the ratio of voids to the volume of u1.

This membrane is made of a hydrophobic polymer.

In the present invention, [hydrophobic J] refers to substantially not getting wet with water. This property often behaves when the polymer thin film has pores, allowing water vapor to pass through but not water or aqueous solutions.

One group of polymers with such hydrophobic properties are fluororesins. Specifically, for example, tetrafluoroethylene, hexylifluorobrobylene, trichlorofluoroethylene, nylidene fluoride, and other fursulfolefins used alone, copolymers of these, and copolymers containing these in predominant amounts. There are copolymers with other olenoins.

Another group of hydrophobic polymers are silicone resins (including rubber). Silicone resins are also known polymers, and an appropriate one may be selected and used.

Methods for producing porous thin films with predetermined pore sizes from these polymers are known, and such porous thin films are commercially available. For example, as a breathable waterproof film, the water osmotic pressure is 0.9/ct to 0.4-25.
i, ventilation ffi/10.016~465 cc/min/c
A commercially available fluororesin material having a d/12 and 4 cm water column pressure is one of the suitable barrier layers for use in the present invention.

In order to attach such a thin film to the alcohol oxidase immobilized membrane of the basic alcohol sensor, the tip of the sensor consisting of the immobilized membrane is coated with a hydrophobic membrane directly or via a cellophane membrane, etc., and then the tip is coated with a hydrophobic membrane using an O-ring or the like. A method may be used in which this membrane is fixed to the tip of the sensor.

As a barrier layer, a porous polytetrafluoroethylene (PTFE) membrane (type Fl-, pore size 0.5 μm, manufactured by Millibore) was used as a barrier layer, and a highly selective alcohol sensor was prepared as follows. alcohol oxidase (Candida boidii origin, Sigma
(manufactured by company a) 50U in 0.1M phosphoric acid buffer? -(pH7
, 8) dissolves in 1.0 m. This was impregnated into an acetyl cellulose membrane (pore size 0.2 μm, manufactured by 1 Millibore), and
The membrane was washed with water for several hours to obtain an immobilized alcohol oxidase membrane. P -r of oxygen electrode (galvanic cell type, Ishikawa Seisakusho)
This membrane is tightly attached to the FE membrane, its surface is covered with a cellophane membrane, and the top is roughly covered with a porous PTFE membrane.
Fixed with a rubber ring. The alcohol sensor thus prepared showed concentration dependence for methanol and ethanol, but did not respond at all to formic acid, acetic acid, and lactic acid.

Table 1 Relative responsiveness of T7 sensor to alcohol and organic acid Solute 8 Highly selective alcohol Alcohol sensor sensor methanol 410 240 ethanol
100 100 formic acid
0 10 acetic acid
0 43 lactic acid
0 32 umbrella thick if: 10
■/liter buffer thickness] 192 Using the alcohol oxidase-immobilized membrane prepared in the same manner as in Example 1, and using silicone rubber as the gas permeable membrane, formic acid, acetic acid and lactic acid did not respond at all.

Example 3 Two types of porous PTF with different pore sizes as a barrier layer
E membrane (Boretex, porosity 89.1%, pore size 6.7
μm111 thickness 0.095m and porosity 89.3%
, with a pore diameter of 5.3 μm and a film thickness of 0.095 s),
When a calibration curve for ethanol was obtained in the same manner as in Example 1, the response amounts to a constant concentration of ethanol were 0.8 times and 0.4 times, respectively, and the measurement range was 1.25 times (0 to 125jI5F/liter). , 2.5 times (0 to 2
50 ■/liter).

1 Porous PTFE membrane, non-porous PTF as barrier layer
Example 1 using E membrane and acetylcellulose membrane
When the response to ethanol (10 η/liter) and the response to a mixed solution of ethanol and acetic acid (10 η/liter) were determined in the same manner as above, it was found that non-porous PTFEIE
! When using an acetylcellulose membrane, no response was obtained to ethanol, and when an acetylcellulose membrane was used, a response was observed to acetic acid. . When a porous PTFE membrane was used, it responded only to ethanol without being interfered with by acetic acid.

Claims (1)

[Scope of Claims] 1. In an alcohol sensor for detecting alcohol in an aqueous solution, which is composed of a physicochemical device and an alcohol oxidase immobilized membrane attached thereto, the alcohol molecule to be measured in the solution is permeated. A hybrid enzyme membrane alcohol sensor, characterized in that a thin film of a hydrophobic polymer having pores with a pore size of 15 μm or less and a porosity of 25% or more is adhered to the alcohol oxidase-immobilized membrane. 2. The hybrid enzyme membrane alcohol sensor according to claim 1, wherein the hydrophobic polymer is a fluororesin. 3. The hybrid enzyme membrane alcohol sensor according to claim 1, wherein the hydrophobic polymer is a silicone resin.