JPS61212300A - Test piece for detecting alcohol having improved shelf stability - Google Patents

Abstract

PURPOSE:The titled test piece capable of determining an alcohol concentrated with an extremely small amount of a specimen solution to be tested, comprising nicotinamide adenine dinucleotide (phosphate), alcohol dehydrogenase, diaphorase, dyestuff, saccharide and carrier. CONSTITUTION:A test consisting of (a) nicotinamide adenine dinucleotide or nicotin amide adenine dinulceotide phosphate, (b) alcohol dehydrogenase, (c) diaphorase, (d) dyestuff, (e) saccharide (cyclodextrin, soluble starch, etc.), and (f) carrier (gelatin, paper, etc.). It shows excellent shelf stability, and colors or decolors a specimen solution to be tested depending upon content of alcohol in the specimen solution even in the case of an extremely small amount of the specimen solution.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to an alcohol detection test piece with excellent storage stability for measuring the concentration of alcohol, especially ethanol, in a solution by an enzymatic method. .

(Prior Art and Problems to be Solved by the Invention) Measurement of alcohol concentration in solutions is frequently performed in the food industry and chemical industry for the purpose of process control. Among alcohols, the measurement of ethanol concentration in particular is a routine measurement item in medical and clinical laboratory departments, and is also an important test item under traffic regulations.

Many chemical methods that have been used to measure alcohol in solutions rely on changes in the volume of the oxidizing agent required to oxidize the alcohol or changes in the color tone of the oxidizing agent. All of these methods suffer from the serious drawback of a lack of specificity because the oxidizing agents used can react with a variety of volatile substances other than alcohols.

For this reason, alcohol analysis methods using gas chromatography and liquid chromatography are available as physicochemical methods that specifically measure only the target substance.
Although these methods were developed in the 1990s, they can accurately quantify and are specific.

Analysis takes a long time, requires specialized and expensive equipment, and the training of human resources who can fully utilize it.

There were problems with immediacy and practicality.

On the other hand, biochemical analysis methods using enzymes generally have superior specificity compared to chemical or physicochemical methods, and have the advantage of not requiring special or large equipment. There is. As a method for measuring alcohol using this method, the reduced form of nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD11) produced by an enzymatic reaction in a solution using alcohol dehydrogenase is measured by absorbance in the ultraviolet region (340 nn+). How to measure (Sukyanto J. Clini Lab and Invest, 358
Page, 1951, by Bonicesen et al.; 5cand,
J, CI in, Lab.

and Invest, + 358+ 1951+ l
? , Bonnichsen at al).

A method in which the formazan dye produced when NADH is oxidized by performing a reaction in an enzyme solution in which diaphorase and a tetrazolium salt are further added to the above system is measured by absorbance in the visible region (British Patent No. 1.351, No. 547) has been proposed. In addition, hydrogen peroxide produced using alcohol oxidase is reacted with orthodianisidine by the action of peroxidase, and its color is changed to 436.
How to do it with +m (Biokimi, Biophysic, Acta 1)
51, pp. 330-342, 1968, Jansen et al.; Biochim.

Biophys, Acta, 151.330-34
2.1968. Frank.

W, Janssen et al) have been reported. Although these methods have excellent specificity, they are both spectroscopic and optical methods, so equipment such as a spectrophotometer is required, and in the case of turbid samples such as blood or food. Measurement was not possible. In addition, in order to carry out an enzyme reaction, it is necessary to dissolve nine different reagents and then dispense them in predetermined amounts into one reactor, which is a complicated operation, and the dissolved reagents have poor storage stability, making it less versatile. Missing.

In order to overcome these drawbacks, methods for measuring alcohol by combining enzymatic methods and electrochemical methods have been studied.
No. 6019 uses alcohol dehydrogenase (alcohol dehydrogenase).

A method is described in which hydrogen produced from ethanol in an enzymatic reaction is electrically measured using an oxidation-reduction system. Although this method allows simple and specific measurement of alcohol, it requires an enzyme-immobilized membrane and an electrical measuring device, and the enzyme membrane also lacks durability.

For example, a liquid enzyme membrane is only stable at 4°C for 2 days, and an enzyme-immobilized membrane is only stable for 2 weeks (which is not practical).

In addition, the amount of sample required for measurement is considered to be a relatively large amount, which is enough to swell the electrode, and there is a limit to the amount of blood that can be collected when used for clinical tests, so this method is also versatile. .

It lacks generality and practicality.

Therefore, there is a need in the industry for a method that is easy to carry, does not require special equipment or techniques, and can easily measure alcohol concentration in a small amount of sample at any location. The current situation is that this has not been the case until today.

However, recently, with the ease of measurement and carrying.

For purposes of speed and accuracy, the said British Patent No. 1.3
No. 51,547 proposes an alcohol measurement composition in which an enzyme-containing measurement solution in a solution state is impregnated into a solid carrier as it is or together with an enzyme activity inhibitor and then freeze-dried ( Japanese Unexamined Patent Publication No. 59-16
6098).

Although this composition has achieved the previous objectives.

Stored in a dark bottle due to insufficient storage stability.

It is necessary to store it under dry conditions in a freezer (-15°C) or a refrigerator, which is impractical.

On the other hand, Japanese Patent Application Laid-Open No. 56-7724 discloses that dextran is coexisting in order to prevent sensitive active ingredients such as pharmaceuticals from being deactivated during the multi-step preparation process such as tabletting, dragee-coating, and capsule filling. , a preparation suitable for pharmaceuticals or diagnostic agents obtained by adopting a freeze-drying method with few steps is described, but it is not disclosed that the test piece is stable or even that alcohol can be measured. It has not been.

(Means for Solving the Problems) As a result of extensive research in order to solve these problems, the present inventors discovered that NAD or NADP, alcohol dehydrogenase, diaphorase, and 9 pigments. , a test piece consisting of saccharides and a carrier showed remarkable storage stability,
Furthermore, the present inventors have discovered that even a very small amount of a test liquid develops or decolors depending on the alcohol content in the test liquid, and the alcohol concentration can be measured, leading to the completion of the present invention.

That is, two aspects of the present invention include (a) NAD or NADP;

(b) alcohol dehydrogenase; (c) diaphorase; (d) dye; (eli); and (f) a carrier.

When the test piece of the present invention is impregnated with a solution containing alcohol, an enzymatic reaction occurs immediately with the alcohol, and the alcohol first reacts with the alcohol in the presence of NAD or NADP.

Aldehyde and NA by alcohol dehydrogenase
Step of conversion to D or NADPH (Reaction formula-1)
9. The reaction consists of a step in which the generated NADH or NADPH transfers hydrogen to the dye by diaphorase, and as a result, the dye fades or develops color (reaction formula-2). The amount of alcohol is measured by immersing a test piece in a solution containing alcohol and visually observing the degree and speed of color fading or color development.

Reaction formula-1 Alcohol dehydrogenase Alcohol reaction formula-2 Diaphorase NAD(P)H ten dyes (oxidized type) NAD(P) ten dyes (reduced type) The alcohol dehydrogenase used in the present invention is generally eight alcohols: NAD oxide
reduce'ase+EC 1, 1, 1.1 and Alc
ohol: NAD (P) oxtdored
uctase.

Any substance that is classified as EC 1, 1, or 1.2 and can dehydrogenate the alcohol to be measured may be used.For example, any substance derived from animal organs or microorganisms can be used. They are already commercially available worldwide from several companies (Boehringer Mannheim Yamanouchi Co., Ltd., Oriental Yeast Co., Ltd.). In addition, when using alcohol dehyde dehydrogenase derived from thermophilic bacteria,
For example, Bacillus stearothermophilus may be produced according to a conventional method.
To purify alcohol dehydrogenase from thermophilus.

Biochemical Journal Volume 127, No. 3 63-64
page. 1972 (Biochem. J. 127
．． 3.63-64. 1972, A. ATKIN
SON) and F. E, be, varnish.

Letter volume 33. No. 1, pages 1-3, 1973 (F.f!
．． B. S. Lett. + 33+1+1~3, 197
3. Bridgegen. John's method can be used.

Also, diaphorase. NADH: lipoami
de oxido-reductase E C 1.
6.4.3, diaphorase NADH: dye o
xidoreductase E C 1.6.99. Any material that can reduce the dye used may be used. This is also derived from animal organs.

Products derived from microorganisms are already commercially available (Beichiringer Mannheim Yamanouchi Co., Ltd., Toyobo Co., Ltd.). Diaphorase derived from thermophilic bacteria can also be used. For example, Bacillus stearothermophilus (Bacillus stearothermophilus)
In order to obtain diaphorase of S. aerothermophilus, Biochemical Journal Vol. 191, 457
~465 pages, 1980 (Biochem, J.,
191.457-465, 1980).

In addition, aldehyde dehydrogenase (
Aldehyde: NAD oxidoreducta
se EC1, 2.1.3. Or Aldehyde:
NADP oxidoreductase EC1.2
．． 1.4. or Aldehyde:NAD(P)
oxidoreductase EC1.2.1.5)
It is preferable to add Any enzyme may be used as long as it can dehydrogenate the aldehyde produced when the alcohol to be measured is dehydrogenated.

Furthermore, NAD and NADP are also available as reagents, for example, from Boehringer Mannheim Yamanouchi. Oriental Yeast Co., Ltd.
Sigma company. A commercially available product from Seikagaku Kogyo Co., Ltd. may be used. Any dye may be used as long as it develops or fades due to the action of diaphorase, such as ferricyanide compounds and what is generally called formazan dye. Potassium ferricyanide, an example of the latter iodonitrotetrazolium salt, can be used. Further, other dyes include methylene blue, 2,6-cyclophenol indophenol, indigo carmine, amaranth, etc., which are dyes whose safety has been confirmed from the viewpoint of safety and health of the tester.

In other words, those that are permitted as food additives.

It is preferable to use indigo carmine, amaranth, and methylene blue, which are used as medicines.

An alcohol detection test piece using a dye that has been confirmed to be safe is ideal for detecting alcohol concentration in saliva, and has great advantages in measuring ethanol under traffic regulations.

Examples of the tRs used in the present invention include oligows such as cyclodextrin and maltotriose, and polyesters such as dextran and soluble starch. In addition, sugar alcohols such as sorbitol, single IR derivatives such as amino sugars such as glucosamine, or two P! derivatives of
Derivatives of oligosaccharides or polyclass Ii derivatives can also be used. These can also be used in combination of two or more types.

Also, as a carrier for use in the test piece of the present invention.

Any substance that is insoluble or poorly soluble in water at room temperature (solubility 1.0% or less, 25°C) is acceptable.

For example, gelatin, agar, cellulose, paper, and chitin.

Low-molecular to high-molecular natural products such as glucan, rayon, vinylon, polyester, certain types of polyvinyl alcohol, and nylon.

Anything from semi-synthetic to synthetic polymers such as acrylics can be used. Any size, thickness, and shape may be used. In preparing the test piece of the present invention, the concentration of each reagent may be determined as follows. For example, when making a test piece for alcohol detection with a concentration of 1 mol/l, the two dyes should be 0.01 to 10 mol/l, preferably 0.1 to 2.
0 mol/1. Most preferably, it is 0.2 to 1.0 mol/l (alcohol dehydrogenase, aldehyde dehydrogenase, and diaphorase are each 10" to 1 mol/l).
QII units/n, preferably 104-108 units/l.

Most preferably 106 to 108 units/! And it is sufficient. Also, NAD and NADP are O, OQ1~2.0
Mol/β.

Preferably it is 0.01 to 1.5 mol/l, most preferably 0.1 to 1.0 mol/l. Sugars are 0.05
It may be at least 0.5% by weight, most preferably at least 2.0% by weight. Any type of buffer can be used to dissolve these.

Its pH is 3-12. Preferably 6-11. Most preferably it is 7.0 to 9.5.

Its concentration is 0.02 to 2.0 mol/j? , preferably 0.01 to 1.0 mol/It, most preferably 0.02 to 0.5 mol/L. An example of such a buffer is a Tris-HCl buffer. When the alcohol concentration to be detected is lower than 1 mol/l, depending on the lower proportion.

It is sufficient to reduce each component, but in reality, it is common to reduce only the 9 dyes because the time required for the enzymatic reaction becomes longer. Also, depending on the nature of the enzyme used, NAD
NADP alone, NADP alone, or both together may be added. It is also possible to add interfacial agents such as Tritomen-100 and Twin-80. The unit of enzyme added is 1 international unit, and 1 unit of substrate is added per minute.
It refers to the activity of changing micromoles under reaction conditions of 25 to 30°C.

To prepare the test piece of the present invention9, for example, a part or all of the water-insoluble or poorly soluble carrier may be impregnated with a solution of each of the above-mentioned reagents, and it may be used as it is after being lifted, or it may be used after drying. , just make it available for use. Alternatively, it may be manufactured by laminating one layer at a time. When using a large carrier, after soaking it in the solution of the above reagent,
It may be cut into pieces of a suitable size, for example, 5 mm in width and 40 mm in length. When drying, it is better to perform low temperature drying or freeze drying.

To actually measure the alcohol concentration in a solution using the test piece of the present invention9 For example, a test piece with a concentration suitable for measurement is immersed in the solution to be measured, pulled out, and left at room temperature for the time required for the dye to fade or develop. This can be done by measuring. Alcohol concentration can also be measured by color change.

When measuring the alcohol concentration in saliva, the alcohol concentration in saliva can be measured by soaking an alcohol detection test piece in saliva and measuring the time required for color fading, or by visually observing a change in color tone.

(Example) Next, the present invention will be specifically explained using examples.

The following reagents were used in this example.

1. NAD or NADP; manufactured by Oriental Yeast Co., Ltd.

2. Alcohol dehydrogenase; Oriental Yeast Co., Ltd. Catalog Fish ADH-01° 3. Diaphorase; Boehringer Mannheim Yamanouchi Co., Ltd. Catalog Dehydrogenase 41155B.

Furthermore, one unit of alcohol dehydrogenase is 25
It is the amount of enzyme that consumes 1 micromole of ethanol per minute at 25°C, and one unit of diaphorase is the amount of enzyme that consumes 1 micromole of NADH per minute at 25°C in the presence of iodonitrotetrazolium violet.

Example 1 Methylene blue 4 mmol/l, <hereinafter abbreviated as mM) Alcohol dehydrogenase 340 units/ml
, diaphorase 100 units/m+, Lydone 0.26mm
), and after freeze-drying, it becomes 5 mm wide and 40 mm long.
A blue test piece (■) was obtained by cutting the sample into strips.

For comparison, a blue test piece (■) was obtained in the same manner as above except that β-cyclodextrin was not used.

The test pieces I and ■ were immersed in a 0.5 g ethanol solution. After taking it out, leave it at room temperature.

When observed with the naked eye, the blue color in the immersed portions of both test pieces began to fade and became colorless in 1 minute. When we measured the time required for color fading by varying the ethanol concentration of the ethanol solution used for dipping, we found that the higher the ethanol concentration, the shorter the time required for color fading.

When the ethanol concentration became very dilute, the color fading did not occur completely and only nine tones became lighter. The fading process was the same for both test pieces, and Table 1 shows the correlation between the change in color tone and the ethanol concentration.

Table 1 As shown in Table 1, the ethanol concentration in the solution can be measured by measuring the time required for color fading and visually observing the change in color. Piece I
There was no difference in the reactivity to ethanol between the test pieces and A decrease in performance was observed in the one piece.

Discoloration completely stopped after one month of storage at 4°C.

on the other hand. The test piece (■) containing cyclodextrin showed no change in performance when stored at 4°C for more than 3 months, and when stored at 37°C for more than 12 days.

The results are shown in Table 2.

Table 2: Validity period of the test piece As shown above, the stability of the test piece was significantly improved by adding cyclodextrin. 2 when stored at 4℃
For over a month. It maintained its performance. In addition, test piece I reacted to ethanol but did not react to methanol. The specificity was as high as when no cyclodextrin was added.

Example 2 Among the compositions shown in Example 1. A test piece (2) was obtained in the same manner as in Example 1, except that cyclodextrin was replaced with dextran (molecular weight: 60,000 to 90,000, 100 g/j!).

The obtained test piece (2) was immersed in solutions with varying ethanol concentrations in the same manner as in Example 1, and the changes were tracked, and results exactly the same as in Example 1 were obtained.

There was no difference in reactivity to ethanol between test pieces ■, ■ and test piece ■.

Next, as in Example 1, we investigated the storage stability of the test piece containing dextran in thermostatic chambers at 4°C and 37°C. However, no deterioration in performance was observed, confirming that the addition of dextran was effective in improving the storage stability of the test pieces.

Example 3 A test piece (2) was prepared in the same manner as in Example 2, except that alcohol dehydrogenase and diaphorase in the composition shown in Example 2 were each replaced with those derived from Bacillus stearothermophilus.

In addition, for comparison, a test piece (2) not containing dextran was also prepared in the same manner.

When the obtained test pieces IV and V were immersed in solutions with varying ethanol concentrations as in Example 1 and the changes were tracked, all the test pieces obtained completely the same results as Example 1, and the process was successful. There was no difference in reactivity to tanol. Test piece V, which does not contain dextran, has excellent storage stability because it uses a stable enzyme derived from thermophilic bacteria, and its performance was maintained even after storage at 37°C for 3 months. In addition, the test piece ■ contains enzymes derived from thermophilic bacteria and dextran.

Furthermore, it has excellent storage stability. There was no change in performance even after 4 to 6 months of storage at 37°C, confirming that the addition of dextran was effective in improving the storage stability of the test piece even when enzymes derived from thermophilic bacteria were used. It was done.

Example 4 Using the test piece (■) obtained in Example 3, the alcohol concentration in saliva was measured.

In other words, 360m of sake for 2 adult men weighing 65kg.
1 hour after drinking, saliva was collected.

When the ethanol concentration in the saliva was measured by gas chromatography (Shimabara Seisakusho GC-3BT), it was found that 0.52 g/l of ethanol was contained.

Next, I applied this saliva to the test piece■.

The dark blue color faded to white in 55 seconds, and from the results of Example 1,
It was found that about 0.5 g/l of ethanol was contained. This result agreed with the result obtained by gas chromatography. In other words, it was confirmed that the ethanol concentration in saliva could be easily measured by using the test piece (■).

(Effect of the invention) The test piece of the present invention has specificity for alcohol.

without compromising characteristics such as the time required for detection.
Storage stability at room temperature is significantly improved, and alcohol concentration can be easily detected at any location and in a short period of time with a small amount of sample.

Therefore, it is very useful in urgent cases such as the diagnosis of acute alcohol poisoning and for use in traffic law enforcement.

Claims (1)

[Claims] (1) (a) nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate; (b) alcohol dehydrogenase;
(c) diaphorase, (d) pigment, (e) saccharide,
(f) A test piece for alcohol detection with excellent storage stability consisting of a carrier.