JPS6013255A - Electrode for detection of mutagen - Google Patents

Abstract

PURPOSE:To obtain an electrode which detects a mutagen as cancerigenic substance handily and accurately depending on the lowering of the H2O2 decomposition activity by mounting a membrane having a copper phthalocyanine or hemin as H2O2 decomposition catalyst on an electrode for detecting oxygen. CONSTITUTION:An aceton solution as membrane forming material such as nitrocellulose, polycarbonate or the like is mixed with copper phthalocyanine or hemin to form an H2O2 decomposition catalyst immobilizing membrane on a glass plate by casting or the like. The membrane 6 thus formed is laminated, for example, on a teflon membrane 4 of an electrode for detecting oxygen made up of a Pb electrode 1, a Pt electrode 2 and a glass tube holding 30% NaOH electrolytic liquid with the membrane 4 on the bottom thereof to build an electrode for detection of mutagen. Then, the electrode thus obtained is immersed into a liquid in which a sample to be inspected (urine or the like) is added to a control liquid 8 comprising a specified amount of H2O2 and a buffer liquid and changes in the current value are measured from that in the condition using the control liquud alone. When any mutagen (e.g. benzo alpha pyrene) exists, the current value decreases according to the amount thereof due to reduction in the H2O2 decomposition capacity thereby enabling the detection with a high sensitivity.

Description

[Detailed description of the invention] The present invention relates to an electrode for mutagen detection.

Today, there is less interest in environmental carcinogens from the viewpoint of preventive medicine, but since there is a high correlation between carcinogens and mutagens, the Ames test is recommended as a primary screening method for carcinogens. Widely used.

However, since this method is complicated and requires a long time of 1 to 2 days, it is not necessarily satisfactory as a simple method for detecting environmental mutagens.

On the other hand, recently, copper phthalocyanine and hemin are benzo(α)
It is becoming clear that it specifically binds to polycyclic mutagens such as pyrene and Trp-p-1 (Yagi, Me.
tal-* Cancer Biochem, Bi
ophys, Rei 1981 Vol, 5. pp, 175
-181; Arimot.

et al., Cancer Letters, 11.
29-33 (] 980); Arimoto et
al,,Biochem,Biophys,Res.

Comm, 92.662-668 (1980) and Kobayashi et al. EMS Japan 11th Conference (October 1982), etc.).

In view of the above, the present inventors conducted extensive research and found that by using copper phthalocyanine or hemin fixed electrodes, the formation of hydrophobic complexes between mutagens and these can be easily amperometrically measured using their H20□ decomposition catalytic activity as an indicator. The present invention was achieved by discovering that accurate detection is possible.

Hereinafter, the structure of the present invention will be explained in detail.

Preparation of catalyst fixed membrane Copper phthalocyanine and/or hemin as a H2O2 decomposition catalyst and a membrane forming material such as nitrocellulose, acetylcellulose, polycarbonate, Nafion, etc. are dissolved and mixed in a solvent such as acetone, and the mixture is cast on a glass plate. Although catalyst-immobilized membranes can be easily prepared by this method, these catalysts may also be directly chemically bonded and immobilized to cellulose membranes or the like by conventional methods.

Here, the film thickness, catalyst concentration, etc. vary depending on the conditions of use, but in general, the film is a concave circle with a film thickness of 0.1 to several tens of micrometers and a catalyst concentration of about 0.1 to several tens of weight coefficients. .

Here, one example of the preparation of a copper phthalocyanine-fixed film will be described in more detail as follows.

Sunawachi, copper phthalocyanine 2 ~ and nitrocellulose 8
A fixed film having a thickness of 5 μm was obtained by dissolving and mixing 8.8 in acetone 2- and casting it into a glass tube to form a film.

Measurement condition It is used as a mutagen detection electrode for measurement.

Here, if you refer to a schematic explanatory diagram (Fig. 1) of one example of a measuring device, pb electricity! 1. PT electrode 2. The identification film 6 is laminated on the Teflon film 4 of the oxygen detection electrode formed of a glass tube 5 consisting of a bottom Teflon film 4 with an electrolyte 3 to detect mutagen. constitutes an electrode for use.

Furthermore, this electrode is inserted into a cell 10 filled with a test liquid 8 having a star 9 and bar 7 placed on a magnetic stirrer 9 so as to be in contact with the test liquid 8 .

The current generated in proportion to the amount of oxygen generated is
It is recorded on the recorder 11 via.

The test solution is prepared by adding the test sample to a control solution consisting of a predetermined amount of H2O2 and a buffer solution, and the mutagen is detected by the decrease in the current value.

The present invention will be explained in more detail below using experimental examples.

Experimental Example 1: Various concentrations of trocellulose (based on 88 kg) were prepared as fixed membranes according to the above copper phthalocyanine fixed membrane preparation example,
0.1M phosphate buffer containing 0.98M H2O22.
Using 0 m (pH 7.0) as a control solution, the current increase value (μA) was measured using the measuring device shown in Figure 1 to check the catalytic activity of the fixed membrane. The results are summarized in Figure 2.

As is clear from the figure, the amount of copper phthalocyanine is 2η/887
It was found that q nitrocellulose had a substantially steady value.

2. Using a fixed membrane of copper phthalocyanine 2η/nitrocellulose 88%, 0.1 M lj phosphate buffer (pH 7)
, 0) The relationship between the amount of H2O2 added (-) and the current increase value (μA) was determined in the same way as above. 3rd result
As shown in the figure, it was found that a substantially steady value was reached at about 0.98 M H2O2.

3. Using the fixed membrane, add 2~6 x 10''-'M of benzo(α)pyrene to 0.1M phosphate buffer (J)H7,0)2 containing 0.98M H2O2. The current reduction value (μA) with respect to the control solution was measured.
A summary is shown in the figure. As is clear from the figure, benzo (α)
There was an extremely good correlation between the amount of pyrene and the current reduction value.

4. In the same manner, the current reduction values (μA) were measured for 1 mM of various organic compounds, and the results are summarized in Table 1 below.

As is clear from the table, the electrode of the present invention is a polycyclic compound.
ylsulfoxide 0n-HexaneO Cyclohexane 0 Tetrahydrofuran 0 Pyridine 0.02 B enzene 0.05 Naphthalene O,07 B enzo (C4pV-rene 6 .45, Similarly, urine of smokers and non-smokers was added to the control solution from 0.2 to 1.
0 m was added and the current reduction value (μA) was measured. The results are summarized in Figure 5.

In the figure, the code a corresponds to smokers and b corresponds to non-smokers).

Furthermore, in order to confirm the correlation with the Ames test, 6
XIF'M benzo (α) pyrene, smoker's bed, and non-smoker's urine were supplied at each Q, 1 mJ, and a control solution of 2.0 ml was applied to determine the current reduction value (μA) and Ames test.

The results are shown in Table 2 below.

Table 2 Benzo(α) Billet 7 5.5+ Smoker's urine 1.3+ Non-smoker's urine 0.4 Control 0-6, Hemin 4'II9, Nitrocellulose 80rIQ Using a fixed membrane made of Example 5 Similarly, benzo (
Ql pyrene, flow reduction value and Am in urine of smokers and non-smokers
As a result of measuring the correlation with es test, a good correlation substantially equivalent to that in Table 2 above was obtained.

[Brief explanation of the drawing]

Figure 1 is a schematic explanatory diagram of a measuring device in an experimental example of the present invention (((), and Figures 2 to 5 are explanatory diagrams of an experiment in the same experimental example. 1...pb electrode, 2... ...PT electrode, 4
...Teflon membrane, 5...Glass tube, 6...Copper phthalocyanine fixed film, and 11.
...Recorder. Special plan applicant Matsunaga Figure 1 □□] Figure 2 Figure 3

Claims (1)

[Claims] (11) A mutagen detection electrode consisting of an oxygen detection electrode attached with a copper phthalocyanine and/or hemin fixed membrane.