JPH022918A - Microorganism detecting method - Google Patents

Abstract

PURPOSE:To make it possible to detect oxidation-reduction current based on bacteria by using a low voltage by using basal-plane pyrolytic graphite (BPG) which is chemically modified with a cobalt II phthalocyanine as a working electrode. CONSTITUTION:BPG 10 which is chemically modified with a cobalt II phthalocyanine is used as a working electrode, and the oxidation-reduction current of microorganism is measured. At this time, the oxidation-reduction current based on bacteria can be detected by using a low voltage by the catalysis of the cobalt II phthalocyanine. Therefore, the detection of the bacteria can be performed readily and highly accurately.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for detecting microorganisms, and particularly to an electrochemical method for detecting microorganisms.

[Conventional technology]

Detection of microorganisms plays an important role in the fields of fermentation industry and clinical testing, but the widely used colony counting method and direct observation method using a microscope are complicated, time consuming, and require skill.

Recently, Matsunaga discovered a phenomenon in which a current is obtained when living cells come into direct contact with an electrode, essentially a basal plane pyrolytic graphite (BPG) electrode, and reported that this can be used for electrochemical identification of cells and a method for controlling activity. (Japanese Patent Application Laid-Open No. 60-114763). Matsunaga et al. found that the current generated by contact between living cells and electrodes is due to the oxidation-reduction of supplementary oxygen A (CoA) in the cells, and cells can be identified by measuring the current generated by electrochemical methods. It is made clear that

[Problem to be solved by the invention] Matsunaga's method can detect microorganisms in a short time with simple operations, but the oxidation of CoA in cells is approximately 0.7 (V vs SC
E (saturated electric current tJi+)), it is susceptible to the influence of coexisting substances, and there are also problems such as the need to clean the surface once every measurement.

The present invention has been made in view of the above points, and its purpose is to provide a method for detecting microorganisms at a more base potential by utilizing a chemically modified BPG electrode.

[Means to solve the problem]

According to the present invention, the above object is to provide an electrochemical microorganism detection method in which cobalt (■) phthalocyanine 111
This is achieved by measuring the redox current of microorganisms using Basalplane Pyrolithink graphite 10, which has been chemically modified with , as a working electrode.

Cobalt (II) phthalocyanine has a planar molecular structure as shown below. The central cobalt atom forms covalent and coordinate bonds with nitrogen.

CoA has a thiol-type molecular structure as shown below.

Nil□ amount Cobalt (■) phthalocyanine can be prepared by spraying or dipping methods.

It is deposited on BPG using a technique such as vapor deposition. C on BPG
oA is electrochemically redoxed. CoA can be analyzed by methods such as amperometry and triangular wave voltammetry.

This is written as R-SH for simplicity.

[Effect]

Cobalt (■) phthalocyanine functions as a catalyst in the electrochemical redox of coenzyme CoA contained in bacteria. This facilitates the detection of redox currents caused by bacteria.

〔Example〕

Next, embodiments of the present invention will be described based on the drawings.

FIG. 1 is a schematic cross-sectional view of a BPG electrode according to an embodiment of the present invention. In Figure 1, 10 is heli-salplane pyrolytic graphite (BPG), and 11 is BPG.
A Kobal 1-(II) phthalocyanine layer is deposited on the surface of the PG, 14 is an insulating resin, 13 is a bonding portion made of silver paste, and 12 is a lead.

The cobalt (■) phthalocyanine layer can be formed as follows. That is, cobalt (n) phthalocyanine powder (manufactured by Kanto Kagaku) is dissolved in pyridine to prepare a saturated solution. BPG electrode (surface area 0.16c+fi) #
Polish with 1500 grit sandpaper and clean using ultrasonic waves. 10 μl of a saturated solution of pyridine containing cobalt (II) phthalocyanine was applied to the BPC and electrodes, left for 1 hour, and air-dried. Using the electrode chemically modified with cobalt (■) phthalocyanine as a working electrode in this way and a measuring circuit as shown in FIG. 2, a cyclic voltamgram can be obtained. In Figure 2, l is a working electrode, 2 is a counter electrode made of a platinum coil, and 3 is a saturated Amagi which serves as a potential reference for the working electrode. ffm (SCE), 5 is a function generator, 4 is a potentiostat, 6 is an x-y recorder, and 7 is a measurement liquid. The measurement temperature is 25°C.

An arbitrary function is generated by the function generator 5, and a predetermined potential is applied via the potentiostat 4 between the working electrode 1, which is a chemically modified BPG electrode, and the saturated agar electrode 3, which is a reference 1 electrode. At this time, the redox current flowing between the working electrode 1 and the counter electrode 2, which are chemically modified electrodes, is measured. The potential sweep is 0 to 0.5 (V vs SCE), and the draw rate is 10 mV/S.

Yeast Saccharomyces cerevisiae
cescerevisiae (I F O0203
) ) is 3% in a CP (glucose peptone) liquid medium (manufactured by Otama Nutritional Chemicals) containing glucose and peptone as main components.
The cells were cultured with shaking at 0°C for 118 hours.

After yeast was collected by centrifugation, it was dispersed in 0.1 M phosphate buffer (p It 7 ) to prepare a measurement solution. The yeast concentration at this time was 4 x 10'' cells/ml.

FIG. 3 shows the cyclic voltagram obtained by JJ.

Here, the potential was swept in the direction of the arrow. Curve A is a blank value in a buffer solution. Curve B is a cyclic voltamgram of a measurement solution containing yeast.

In the voltagram of a liquid containing yeast, an inflection point in the oxidation direction is observed at about 0.26V, and an inflection point in the reduction direction is observed at about 0.14V, and oxidation waves and reduction waves due to yeast are observed. The difference in current value between the test solution containing yeast and the blank solution is 0.16 μA at 0.4 V for the oxidation reaction and 0.06 V for the reduction reaction.
It is 0.15μA. In addition? ii? For R, a positive value indicates a reduction current, and a negative value indicates an oxidation current.

Next, when the yeast concentration was changed to 1×1O8 cells/ml and the yeast concentration was constant, the difference in current value between the blank current at 0.06 V and 0.4 V was 0.04 μA. This fact indicates that the current obtained is proportional to the yeast concentration.

Furthermore, when all determinations were performed five times in succession using a measurement solution with a yeast concentration of 4×10 8 cells/d, the obtained current values matched within measurement error. This shows that surface treatment is not necessary for each measurement.

When coenzyme A is determined atlI under the same conditions as in Figure 3, the inflection point in the oxidation direction is approximately 0.22 V, and the inflection point in the reduction direction is approximately 0.12 V. It can be seen that it shows a redox potential.

By using chemically modified BPG as a working electrode, the reversibility of the reaction increases and the oxidation reaction occurs at a more base potential, eliminating interfering components and increasing the accuracy of bacterial detection. can. Furthermore, if a chemically modified BPG electrode is used, it becomes possible to detect bacteria not only by the above-mentioned oxidation waves but also by reduction waves. Furthermore, as a result of surface modification, oxidation occurs at a base potential, resulting in surface oxidation that causes a decrease in scratching activity, and the precipitation of coexisting substances such as nitrite ions and oxalate ions, which are found in fermentation samples containing bacteria, on the electrode surface. can be reduced and the stability of the electrode can be improved.

〔Effect of the invention〕

According to this invention, in an electrochemical method for detecting microorganisms, the redox current of microorganisms is measured using basal plain pyrolytic graphite chemically modified with a cobalt (II) phthalocyanine layer as a working electrode. Due to the catalytic action of the oxidation-reduction current, which is effective against bacteria, it is possible to detect the redox current using a low voltage, which makes it possible to detect bacteria easily and with high precision.

[Brief explanation of the drawing]

FIG. 1 shows a chemically modified 10 according to an embodiment of the present invention.
- B1) G, 11... Cobalt (II) phthalocyanine. Tomi 1 Figure 0.1 0.2 0.3 0.4 0.5 6ρ applied voltage (V blade S(,E)

Claims (1)

[Claims] 1) An electrochemical method for detecting microorganisms, characterized in that the redox current of microorganisms is measured using basal plain pyrolytic graphite chemically modified with a cobalt (II) phthalocyanine layer as a working electrode.