JPH0470558A - Ethanol sensor - Google Patents

Abstract

PURPOSE:To easily form an electrode and to carry out measurement within a short time by using a hydrophobic compound as an electron acceptor and combining acetic bacteria membrane bonding alcohol dehydrogenase, an electron acceptor modifying electrode and a reference electrode. CONSTITUTION:An electron acceptor composed of a hydrophobic compound synthesized from water-soluble ferricyanate and a cationic surfactant, an amphoteric surfactant or a nonionic surfactant, acetic bacteria membrane bonding alcohol dehydrogenase, a conductive powder and an org. binder are mixed to prepare paste. This paste is applied to the end surface of a graphite solidified body 13 and cured to form an electrode agent 10 to prepare an enzyme and electron acceptor modifying electrode 1. This electrode 1 and a reference electrode 2 are combined to constitute a sensor. This sensor can directly measure a generated oxidizing current amperometrically and has a rapid reaction time and excellent linearity. Further, the electron acceptor can be simply synthesized and isolated in an aqueous solution and this electron acceptor and enzyme are merely mixed with the org. binder to be fixed to the electrode agent.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to an ethanol sensor for measuring ethanol concentration in a liquid sample.

[Conventional technology]

As a sensor for measuring ethanol in a bath liquid, a device is known in which a carrier gas is passed through a gas permeable tube and the ethanol that has permeated into the tube is detected using a gas chromatograph, an infrared gas detector, or the like. Although this method allows on-line measurement, it has the disadvantage that ethanol measurement cannot be easily performed because the device is large and complicated. In contrast, small-sized alcohol sensors that utilize various enzymes that act on alcohol have been developed. These are in practical use because they have a simple configuration and can be produced relatively easily. Among these, those using alcohol oxidase have the disadvantage that the enzyme has low substrate specificity and reacts with methanol in the same way as ethanol. Also, nicotinamide adenine nucleotide (NAD)
Sensors using dependent alcohol dehydrogenase have excellent specificity in that they do not react with methanol, but they require the use of expensive NAD;
It was also difficult to regenerate NAD from NAD, which made it impractical. In order to overcome these shortcomings, a sensor utilizing cell membrane-bound alcohol dehydrogenase (ADH) present in acetic acid bacteria has been developed. This enzyme selectively reacts with ethanol through the involvement of the coenzyme pyrroloquinoline quinone (PQQ). Since PQQ strongly binds to ADH, it does not require special immobilization and has the excellent feature that it does not require the use of NAD. Conventionally, published patents related to sensors using membrane-bound ADH present in acetic acid bacteria include: Japanese Patent Application Laid-Open No. 61-265
No. 560, ■Japanese Patent Publication No. 61-281961, ■Japanese Patent Publication No. 6
2-277548, (1) Japanese Patent Application Publication No. 1-96552, and (2) Japanese Patent Application Publication No. 1-97851. Among these, ■, ■, and ■ do not use an electron acceptor in the measurement system. Therefore, when ethanol is oxidized by the enzyme, the system that returns PQQ, which has changed to the reduced form, to the oxidized form is weak, so it takes time to respond. It takes. (2) and (2) have an oxidized electron acceptor dissolved in the measurement solution. Therefore, during measurement, the electron acceptor is coupled to the enzymatic reaction,
It returns PQQ to its oxidized form and is itself reduced. Furthermore, since this is oxidized using an electrode, measurements can be made in a short time.

[Problem to be solved by the invention]

In above ■ and ■, potassium ferricyanide and phenazine methosulfate (PMS) are used as electron acceptors.
) etc. are used by dissolving them in the measurement solution or electrolyte. This measurement liquid reaches a considerable amount when the sensor is used for a long time. In particular, the flow cell type measurement method requires a large amount of liquid. Utilizing the electron acceptor by dissolving it in the measurement solution or electrolyte at that time has poor workability, and when potassium ferricyanide is used, there arises the problem of cyanide ion waste solution treatment. Furthermore, when using PMS'&, the running cost becomes particularly high. Further, as for the measurement time, (1) requires several minutes, (2) requires 1 to 2 minutes, and when measuring many samples, it is desirable to be able to measure in an even shorter time.

[Means to solve the problem]

In view of the above points, the present inventors have conducted extensive research and have developed an ethanol sensor that eliminates electron acceptors from the electrolytic solution and can perform measurements in a short time. That is, the present invention utilizes a hydrophobic compound synthesized from a water-soluble ferricyanate and a cationic surfactant, an amphoteric surfactant, or a nonionic surfactant as an electron acceptor to produce acetic acid bacteria membrane-bound alcohol dehydrogenase. This is an ethanol sensor consisting of a reference electrode and an enzyme- and electrode-receptor-modified electrode mixed together as an electrode agent. In the present invention, water-soluble ferricyanates include potassium ferricyanide, sodium ferricyanide, and water-soluble ferricyanides thereof. Cationic surfactants include long-chain quaternary ammonium salts and long-chain alkylpyridinium salts, amphoteric surfactants include, for example, alkyldiaminoethylglycine types, and nonionic surfactants include, for example. Examples include polyoxyethylene-based ones. A water-soluble ferricyanate, such as potassium ferricyanide, and these surfactants react in an aqueous solution to produce a yellow precipitate. This precipitated material is used as an electron acceptor in the present invention. An example of the reaction formula is shown below. 3[C+5H3)N(CI*)x]Br +KJe(
CN)a→[C+5t(zz(CHs)zlz[Fe(
CN)a]' + 3KBr Since the obtained compound is hydrophobic, it can be easily purified by filtration and washing with water. In the past, there have been no examples of using such a compound as an electron acceptor in a sensor. In addition, the acetic acid bacteria that produce cell membrane-bound ADH used in the present invention include Acetobacter spp.
bacter) Gluconobacter genus (Glueo
The bacterial cells of any acetic acid bacteria belonging to the genus P. nobaeter may be used. Enzymes can be obtained by separating a cell membrane fraction using a conventional method such as a French press method or an ultrasonic method, further extracting the cell membrane fraction from the cell membrane fraction using a surfactant, and purifying and isolating the enzyme using various types of chromatography. In addition, even crude specimens such as acetic acid bacterium cells as they are or cell membrane fractions can be used as an enzyme source without purification and isolation. In the case of the present invention, as in the conventional method, it is preferable to mix conductive material powder such as graphite with the electrode material so that the current obtained from the surface of the ethanol sensor can be easily guided to the electrode rod. Furthermore, it is preferable to mix an organic binder, such as liquid paraffin, into the electrode material of the ethanol sensor of the present invention, because the enzyme and electron acceptor come into contact with the ethanol-containing sample solution during measurement, and the ethanol-containing sample solution flows out. This is to prevent the electrode material from collapsing and to ensure that the electrode material is modified by adhesion to the electrode surface. To manufacture a modified electrode, first, the ADH-containing material and electron acceptor obtained as described above are thoroughly mixed with a conductive material powder (e.g., graphite powder) and an organic binder (e.g., liquid paraffin) to form a paste. Knead it into a shape. next,
As shown in FIG. 1, the paste is applied to the end face of the graphite solidified material 13 and cured to form the electrode material 10, so that the enzyme and electron acceptor containing the membrane-bound ADH and the electron acceptor in the electrode material are prepared. The body modification electrode 1 can be manufactured. The modified electrode 1 consists of a Teflon cylinder body 15 with a flange.
a copper threaded bolt 12 with a protruding head screwed into it, a graphite solidified material 13 attached to the front part thereof, and further the electrode material 10 joined to the front end surface thereof.
It consists of. Note that a mounting nut 14 is screwed into the middle of the copper threaded bolt 12. Next, as shown in FIG. 2, the above-mentioned modified electrode 1 is placed in a reaction cell 3 and placed one inch in length. A reference electrode 2 is further installed in the reaction cell 3 so that a detection sensor is located therein, and a magnetic stirrer 4 is rotatably supported in the center of the reaction cell 3. A sample supply port 33 is provided at the top, an electrolyte inlet 31 is provided at the lower left end, and a measurement waste liquid outlet 32 is provided at the upper right end. To measure the ethanol concentration in a sample, an ethanol concentration measuring device whose schematic diagram is illustrated in FIG. 3 is used. That is, the measuring electrolyte (for example, pH adjustment buffer and electrolyte (for example, KCI) in the electrolyte tank 6 of the measuring device is
) is injected into the reaction cell 3 using the pump 8, and electricity is applied between the modified electrode 1 and the reference electrode 2 from the constant voltage power supply section of the measuring device. As a result, the oxidation current generated between the two electrodes is converted into a voltage value using a current-voltage conversion circuit, and then amplified and recorded on a chart using a recorder. Note that the amplified voltage value can be A/D converted and then processed by a computer. In Figure 1, 7 is a measured waste liquid tank. After the voltage is stabilized, a certain amount of the aqueous sample solution containing ethanol is injected into the reaction cell 3 using a microsyringe, and the oxidation current is measured as a voltage in the same manner as above to determine the amount of increase in voltage. The ethanol concentration can be determined from the fact that this increase is proportional to the ethanol concentration. During measurement, if the stirrer 5 is started and the magnetic stirrer 45 in the reaction cell 3 is rotated, the sample and electrolyte can be mixed uniformly, so accurate measurement values without variations can be obtained. . By the way, the pH of the electrolyte is adjusted to allow the enzymatic reaction to proceed smoothly.
It is desirable to prepare between 4 and 7, and it is necessary to contain a strong electrolyte such as KCI. moreover,
Measurement can also be performed by a flow injection method in which the electrolytic solution is injected at a constant flow rate into the inlet 31 of the reaction cell and measured while being discharged from the outlet 32. According to the ethanol sensor described above, it is possible to directly amperometrically measure the oxidation reaction of ethanol by the cell membrane-bound ADH of acetic acid bacteria and the oxidation current generated by the electron transport system via PQQ and electron acceptors. Therefore, it opens quickly during response and has excellent linearity. Furthermore, the electron acceptor according to the present invention can be easily synthesized and isolated in an aqueous solution. In addition, since both the electron acceptor and enzyme are hydrophobic, they can be immobilized as an electrode material simply by mixing with an organic binder without any special treatment, and will not be eluted into the electrolyte for measurement. Therefore, compared to other biosensors that have enzyme-immobilized membranes, electrodes are easier to create. The electron acceptor according to the present invention is also modified, which is novel. When performing measurements using the ethanol sensor of the present invention, there is no need to dissolve an electron acceptor such as potassium ferricyanide in the electrolyte for measurement, which improves workability.
There is no need to treat wastewater such as cyanide. Note that it is also possible to widen the quantification range by using reaction cells of various sizes or by adopting a flow injection method.

【Example】

Next, the present invention will be specifically explained using examples. 1 person ADH cultured G Iuconobacter 5obox
ydans (IFO12528>) were collected by centrifugation, and the obtained cells were crushed using a French press.
The precipitate (membrane fraction) obtained by 60 minutes) was used as it was as membrane-bound ADH. 2) - Add 0.05% of n-hexadecyltrimethylammonium bromide, a type of cationic surfactant, to an O, 1M aqueous solution.
Add the same amount of M potassium ferricyanide water bath solution. A reaction occurred instantly, and a yellow-green hydrophobic substance was produced in suspension. This compound was filtered through filter paper, thoroughly washed with water, dried under reduced pressure, and used as an electron acceptor. This material was insoluble in water and slightly soluble in the organic liquid phase. 3> ADH-containing membrane fraction (A) obtained in (1) above of Enzyme Bidecoration, above (
Electron acceptor (B) obtained in 2>, graphite powder (
C) and liquid paraffin (D>, A:B:C:D
= 10:1:12:10 ratio (weight ratio), knead it into a paste, and apply a small amount of it to the tip of the graphite solidified material 13 on the pole as shown in Figure 1 to solidify it. After drying, rub the surface with parchment paper to make it smooth.
A modified electrode 1 containing an enzyme and an electron acceptor was manufactured. (a) Preparation of ethanol concentration calibration curve for 4-enol: The modified electrode 1 manufactured in (3) above was attached to the reaction cell 3 as shown in FIG. 1M phosphate buffer (pH
An electrolytic solution containing O, 1M potassium chloride, and 20M magnesium chloride is injected into the reaction cell 3 through the inlet 31 using the pump 8, and the stirrer 5 is started to rotate the magnetic stirrer 4. and stirred. Next, +0.6 V is applied to the modified electrode 1 and the reference electrode (silver chloride electrode) 2 from the constant voltage power supply, and the current generated between the two electrodes is converted into voltage by the voltage-current conversion circuit. was amplified and recorded on a chart using a recorder. When the voltage becomes stable, the voltage value ■. Next, add a known concentration of an aqueous ethanol sample 'fL5μm to reaction cell 3.
I injected it with a microsyringe. The voltage changes immediately with the addition of the ethanol sample aqueous solution, and the output stabilizes in 30 to 60 seconds, so measure the voltage value (2) at this time and read it as V. The difference ΔV between and V was calculated. Furthermore, the pump 8 pumps the electrolyte into the reaction cell 3 to the cell capacity of 5
After injecting about double the amount and cleaning the inside of the cell 3, the V was applied again. was measured. Baseline return after washing with electrolyte is also 30
It was ~60 seconds. In this way, cell cleaning, voltage measurement,
Repeat the operations of sample injection and voltage measurement for various concentrations of ethanol. (2) was measured and a calibration curve shown in FIG. 4 was created. (b) Measuring the ethanol concentration of the sample: For the fermented liquid of alcoholic fermentation and beer, the MU was measured in exactly the same manner as in (a), and the ethanol concentration was calculated by comparing it with the previously prepared calibration curve. This value was in good agreement with the value measured by gas chromatography.

【Effect of the invention】

As described above, according to the ethanol sensor of the present invention,
The oxidation reaction of ethanol by cell membrane-bound ADH of acetic acid bacteria and the oxidation current generated by the electron transport chain via PQQ and electron acceptors can be directly measured amperometrically, so the reaction time is fast and linearity is excellent. There is. Further, the electron acceptor according to the present invention can be easily synthesized and isolated in an aqueous solution, and since both the electron acceptor and the enzyme are hydrophobic, an organic binder can be used without any special treatment. As soon as it is mixed with the electrolyte, it is immobilized as an electrode agent and will not be eluted into the electrolyte for measurement. Therefore, it is easier to create an electrode than a conventional ethanol sensor having an enzyme-immobilized membrane. Furthermore, when measuring using the ethanol sensor of the present invention, there is no need to dissolve electron acceptors such as potassium ferricyanide in the electrolyte for measurement, which improves workability and makes it easier to treat wastewater such as cyanide. There's no need to.

[Brief explanation of drawings]

Figure 1 is a structural diagram of an enzyme- and electron acceptor-modified electrode according to an example of the present invention, Figure 2 is a structural diagram of a reaction cell, Figure 3 is a schematic diagram of an ethanol concentration measuring device, and Figure 4 is a diagram using an ethanol sensor. The calibration curves for each are shown. In the figure, 1: modified electrode, 2: reference electrode, 3. reaction cell 4: magnetic stirrer 25: stirrer, 6: electrolytic cell. 7: Drainage tank, 8: Bomb, 10: Electrode material. Teflon cylinder with 11:7 langes. 12: Copper threaded bolt. 13. Graphite solids, 14: Nuts. 31・Electrolyte inlet, 32: Measurement solution outlet 33・Sample supply port

Claims (4)

[Claims] (1) Water-soluble ferricyanate and cationic surfactant,
An enzyme and electrode acceptor-modified electrode in which a hydrophobic compound synthesized from an amphoteric surfactant or a nonionic surfactant is used as an electron acceptor and is mixed with acetic acid bacteria membrane-bound alcohol dehydrogenase as an electrode agent, and a reference electrode. An ethanol sensor characterized by a combination of. (2) The ethanol sensor according to claim 1, wherein the electrode material is a mixture of conductive material powder. (3) The ethanol sensor according to claim 1 or 2, wherein the water-soluble ferricyanate is potassium ferricyanide. (4) The ethanol sensor according to any one of claims 1 to 3, wherein the electrode material is made of a mixture of an enzyme, an electron acceptor, and an organic binder.