JP2003075389A - Test piece for quantitative determination of alcohol, and method of determining alcohol quantitatively - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly and simply determine an alcohol concentration quantitatively. SOLUTION: An electrode system including a measuring electrode and a counter electrode is provided on an insulative substrate, and a reaction layer is provided on the electrode system or in the vicinity thereof. The reaction layer contains at least an alcohol dehydrogenase, β-nicotinamide dinucleotide and an electron acceptor. A sample solution is supplied to this test piece of the present invention, a fixed voltage is impressed thereafter between the measuring electrode and the counter electrode after lapse of a prescribed time, and an alcohol concentration is found, based on an oxydation current value flowing between the both electrodes therein.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test piece for alcohol quantification constructed so as to be able to measure a wide concentration range, and a method for quantifying alcohol using the test piece.

[0002]

2. Description of the Related Art Conventionally, for quantifying alcohol such as ethanol and methanol, a vibration densitometer (Alcomate manufactured by Riken Keiki Co., Ltd.) or a quantitative analysis kit by colorimetric method (F manufactured by Boehringer Mannheim Co., Ltd.) -There is a kit). The former can measure the alcohol concentration in about 30 seconds from the vibration generated by heating after placing the sample in the measurement system. The latter kit obtains the amount of β-NADH produced when β-NAD and alcohol react with alcohol dehydrogenase from the absorbance, and converts this into the alcohol concentration. It takes about 30 minutes to complete all the measurements.

[0003]

In the conventional vibration density meter as described above, the device is large and expensive. Also,
The above-mentioned quantitative analysis kit has been so quick that one measurement takes about 30 minutes. All the measurement methods also required pretreatment of the sample solution such as filtration and centrifugation, and the operation was complicated.

[0004]

Means for Solving the Problems In order to solve the above problems, a test piece for alcohol determination of the present invention comprises an electrode system formed on an insulative substrate and comprising at least a measuring electrode and a counter electrode, or on the electrode system or The reaction layer is formed in the vicinity thereof, and the reaction layer contains at least the enzyme alcohol dehydrogenase, the coenzyme β-nicotinamide dinucleotide, and the electron acceptor potassium ferricyanide. The reaction layer preferably further contains the enzyme dihydrolipoamide dehydrogenase. The present invention is a method for quantifying alcohol using the above-mentioned alcohol quantification test piece, which comprises supplying a sample solution to the test piece, and after a certain period of time, a positive measurement electrode between the measurement electrode and the counter electrode. There is provided a method for quantifying alcohol, in which the alcohol concentration is calculated from the oxidation current value flowing between both electrodes by applying the constant voltage.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION When a sample solution, for example, a solution containing ethanol is supplied to the reaction layer of the test piece of the present invention, the reaction layer dissolves in the sample solution, and the ethanol in the sample solution becomes an enzyme alcohol dehydrogenase (hereinafter referred to as ADH). (Represented) to produce acetaldehyde. The coenzyme β-nicotinamide dinucleotide (hereinafter referred to as β-NAD) is reduced by the electrons transferred by the oxidation reaction by ADH, and reduced β-NAD (hereinafter referred to as β-NADH) is generated. Then, β-NADH and potassium ferrocyanide are produced by the reaction between β-NADH and the electron acceptor potassium ferricyanide. Next, by applying a voltage with a positive measurement pole between the measurement pole and the counter pole, a current for oxidizing the generated potassium ferrocyanide is obtained. Since this current value corresponds to the concentration of ethanol that is the substrate, the concentration of alcohol in the sample solution can be determined. In the reaction layer,
Further, when the enzyme dihydrolipoamide dehydrogenase is included, the reaction between β-NADH and potassium ferrocyanide is promoted, so that a highly sensitive alcohol test piece can be obtained. As described above, according to the present invention, the alcohol concentration in the sample liquid can be measured accurately, quickly, and easily.

[0006]

EXAMPLES The present invention will be described in more detail with reference to specific examples. << Example 1 >> FIG. 1 is an exploded perspective view of a test piece for alcohol measurement in an example of the present invention, excluding the reaction layer, and FIG. 2 is a longitudinal sectional view thereof. A method for producing this alcohol test piece will be described. A silver paste is printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate to form silver leads 2 and 3. Then, an electrode system including the measurement electrode 4 and the counter electrode 5 is formed by a conductive carbon paste containing a resin and a binder by a printing method, and an insulating layer 6 is formed by an insulating paste.
The insulating layer 6 has an area of the exposed portion of the measuring electrode 4, for example, about 1 m.
m ² is kept constant and the silver leads 2 and 3 are partially covered.

Next, 0.5 of hydrophilic polymer carboxymethyl cellulose (hereinafter abbreviated as CMC) was applied on the above electrode system.
A wt% aqueous solution is dropped and dried to form a CMC layer. Subsequently, on the CMC layer, 4 μl of a mixed solution of 750 units (U) of the enzyme ADH, 35 mg of the coenzyme β-NAD and 66 mg of potassium ferricyanide as an electron acceptor in 1 ml of deionized water was dropped, and 50 ℃
The reaction layer 7 is formed by drying for 10 minutes in the warm air dryer. The outer peripheral portion of the reaction layer 7 substantially matches the diameter of the counter electrode.

After the reaction layer 7 is formed, the spacer 8 having the slit 9 and the cover 10 having the air hole 11 are adhered in a positional relationship as shown by a chain line in FIG. In the test piece thus assembled, the sample liquid supply path 12 is formed between the substrate 1 and the cover 10 by the slit 9 of the spacer 8. The sample solution is easily introduced into the reaction layer portion by the capillary action of the sample solution supply passage 12 by a simple operation of bringing the sample solution into contact with the sample supply port 12a at the tip of the test piece. The supply amount of the sample liquid depends on the space volume of the sample liquid supply passage, and therefore does not need to be quantified in advance. Evaporation of the sample liquid in the sample liquid supply passage is minimized, and highly accurate measurement is possible.

An ethanol aqueous solution is used as a sample solution on the alcohol test piece prepared as described above.
When supplied from 2a, the sample solution quickly reached the air holes 11 in the sample solution supply passage 12 by the capillary phenomenon, and the reaction layer 7 on the electrode system was dissolved. 11 after supplying the sample
After 5 seconds, a pulse voltage of +0.5 V was applied to the measurement electrode 4 in the anode direction with reference to the counter electrode 5 of the electrode system, and the current value after 5 seconds was measured. The concentration of ethanol in the sample solution was 0.5. The response current value depending on the ethanol concentration was obtained up to%.

When the reaction layer 7 is dissolved in the sample solution, ethanol in the sample solution is oxidized by ADH and acetaldehyde is produced. Β-NAD is reduced by the electrons transferred by the oxidation reaction of ADH, and β-NADH is produced. Then, β-NAD and potassium ferrocyanide are produced by the reaction of β-NADH and potassium ferricyanide. Next, by applying the above-mentioned pulse voltage, a current for oxidizing the formed potassium ferrocyanide is obtained, and this current value corresponds to the concentration of ethanol as a substrate.

Example 2 To the reaction layer of Example 1, 0.3 U of dihydrolipoamide dehydrogenase (also called diaphorase) was added per test piece. According to this example, the reaction of β-NADH and potassium ferrocyanide was promoted by the action of diaphorase. As a result, the sensitivity was doubled. Further, it became possible to shorten the time required to apply a voltage between the electrodes after supplying the sample to 55 seconds. As the electron acceptor, potassium ferricyanide shown in the above examples is excellent in terms of stability and reaction rate, but is not limited thereto, and p-benzoquinone, ferrocene and the like can also be used. is there.

[0012]

As described above, according to the present invention, a sample liquid containing alcohol can be easily measured in a short time without pretreatment.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a test piece for alcohol determination in one example of the present invention, excluding a reaction layer.

FIG. 2 is a vertical sectional view of the test piece.

[Explanation of symbols]

1 Insulating substrate A few leads 4 measuring poles 5 opposite poles 6 insulating layers 7 Reaction layer 8 spacers 9 slits 10 covers 11 air holes 12 Sample liquid supply path 12a Sample supply port

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Shiro Nankai             1006 Kadoma, Kadoma-shi, Osaka Matsushita Electric             Sangyo Co., Ltd. F-term (reference) 4B063 QA01 QQ24 QQ64 QR04 QR65                       QR84 QS39 QX05

Claims (3)

[Claims] 1. An electrode system formed on an insulating substrate, comprising at least a measurement electrode and a counter electrode, and a reaction layer formed on or near the electrode system, wherein the reaction layer comprises at least the enzyme alcohol dehydrogenase A test piece for alcohol determination, which comprises the enzyme β-nicotinamide dinucleotide and an electron acceptor. 2. The test piece for alcohol determination according to claim 1, wherein the reaction layer further contains an enzyme dihydrolipoamide dehydrogenase. 3. A method for quantifying alcohol using the alcohol quantification test piece according to claim 1 or 2, wherein after a certain period of time has elapsed after supplying the sample solution to the test piece, a measurement electrode and a counter electrode were formed. A method for quantifying alcohol in which a constant voltage is applied between the electrodes and the alcohol concentration is calculated from the oxidation current value flowing between both electrodes.