JPH0634005B2 - Electrochemical identification of cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for electrochemically discriminating various cells, and more particularly to a novel method for counting cells such as microorganisms and animals and plants, and a novel method for counting the types of cells. Regarding the determination method.

[Problems to be Solved by Prior Art and Invention]

The taxonomic identification or identification of microorganisms and the like occupies an extremely important position in a wide variety of industrial fields including the clinical laboratory field, but the implementation uses a so-called selective medium. Since it depends on the colony counting method, the direct observation method with a microscope, etc., it is inevitably complicated and requires a long time.

On the other hand, the present inventors previously discovered a phenomenon in which a current is obtained when a living cell directly contacts the electrode, and proposed an electrochemical cell count method utilizing this phenomenon [Analytica Chemika Actor, Anal. Chim. Acta, 98, 25 (1978); Applied and Environmental Microbiology, Appl. Environ. Microbiol., 37, 117 (1979); and European Journal of Applied Microbiology and Biotechnology, Eu.
r. J. Appl. Microbiol. Biotechnol., 10, 125 (198
0)], but the type of cells, the identification of mycological properties, and the mechanism thereof have not been clarified at all.

In view of the above, as a result of further intensive studies by the present inventors, the electric current obtained by contact between the cell and the working electrode is mainly between the active substance such as coenzyme A or the like present in the cell wall of the cell and the electrode. Applying a scanning potential to cells according to the technique of electron transfer and differential pulse voltammetry, differential pulse polarography, phase difference discrimination AC polarography, rectangular wave polarography, etc., including so-called cyclic voltammetry. However, it was found that the cell type can be identified with extremely high accuracy by measuring the generated current value or the differential current value.

Further, according to a method such as differential pulse voltammetry, differential pulse polarography, phase-difference discrimination alternating current polarography, rectangular wave polarography, etc., an increasing scanning potential and an appropriate minute potential superimposed on it are applied to the cells, 4, 4 ′
-It was found that the cell type can be discriminated with extremely high accuracy by measuring the differential current value generated by being enhanced by bipyridine.

That is, a current-potential curve (voltammogram) or a differential current-potential curve (differential pulse voltammogram) obtained by the above method, or a potential value (peak potential value) that gives an extreme value of a current value or a differential current value, or a curve Since the patterns and the like differ from each other depending on the type of cells, it becomes possible to achieve distinction and immobilization of each cell (microorganism) very clearly. The above-mentioned peak potential value is obtained as a half-wave potential value by various polarographic methods.

In particular, in the identification method of the present invention, when cells or the like are densely accumulated on a support such as a membrane filter and brought into contact with the working electrode, an enhanced current value or differential current value is observed. For example, even when a measurement target is a cell-diluted suspension or the like, a clear and distinct identification method is provided by filtering this and accumulating it on a support (filter material) for measurement.

[Means for Solving the Problems]

Therefore, in the present invention, cells or cell wall pieces accumulated on a support are brought into contact with the working electrode in a liquid in which the working electrode and the counter electrode are immersed, and a scanning potential is applied to the working electrode to determine the scanning potential. The present invention relates to a method for electrochemically identifying a cell, which includes a step of measuring a current value or a differential current value generated by application.

Hereinafter, the configuration and the like of the present invention will be described in more detail.

Targets for measurement or control Almost all cells such as bacteria, actinomycetes, molds, microalgae, various microorganisms such as yeasts, erythrocytes, leukocytes, tumor cells, and various animal and plant cells such as cultured animal and plant cells can be identified or controlled. obtain.

Further, in the method of the present invention, not only the living cells are used as they are, but also cells released from an eluate obtained by subjecting the living cells to ultrasonic destruction, mainly cell walls may be used.

Identification or determination of cells By applying a scanning potential to the working electrode by voltammetry and the like, and measuring the current value or the differential current value generated by the application of the scanning potential, the peak potential value or current between the cell and the electrode- The shape of the potential curve or the differential current-potential curve is due to the characteristics of the cell wall or cell membrane of the cell used for the measurement.

For example, as shown in Examples below, Bacillus subtilis, Lactobachillus fermentum, Streptococcus sanguis, and Staphylococcus epidermidis.
peak potential value in Gram-positive bacteria such as midis),
E. coli having a current-potential curve or a differential current-potential curve and a cell wall structure more complicated than that of the Gram-positive bacterium (Escher
ichia coli) and Salmonella typhimurium (Salm
There is a clear difference between the peak potential value and the current-potential curve or the differential current-potential curve in Gram-negative bacteria such as onellatyphymurium).

Therefore, according to the method of the present invention, identification of a wide variety of cells,
For example, bacteriological discrimination of various cells, identification, specifically, discrimination of Gram-negative positive bacteria, discrimination of mutation reversion or non-reverted strain in so-called Ames test, etc. For example, it is possible to make a very wide range of identification.

Various Measuring Conditions and Fields of Application As an apparatus, various ordinary voltammetry apparatuses can be used. A schematic explanatory view of one example of the cyclic voltammetry apparatus is shown in FIG.

That is, the illustrated device is an electrolysis cell 4 having a working electrode 1, a counter electrode 2 and a reference electrode 3 such as a saturated sodium chloride sweet potato electrode (hereinafter referred to as SSCE) and a saturated potassium chloride sweet potato electrode (hereinafter referred to as SCE), and a potentiometer. It comprises an ostat 5, a linear scanning or sweeping power source 6 and an XY recorder or synchroscope 7.

Here, as the working electrode 1 and the counter electrode 2, ordinary platinum, gold, silver, mercury, carbon, etc. electrodes and various modified electrodes such as coating these with a conductive polymer can be used.
If the potential of the counter electrode 2 is stable and invariable, a circuit configuration equivalent to that of a normal polarography lacking the reference electrode 3 is sufficient.

As for the electrolysis cell, as a specific example of the present invention, as shown in FIG. 3, a working electrode (105) in contact with a support or a filter (108) holding cells (107) to be measured, An example is an electrode system in which a case (109) made of plastic or the like to support this, a counter electrode (104) and a reference electrode (106) partitioned by a glass tube are immersed in a phosphate buffer solution.

As the support or filter for holding the cells, various synthetic resin porous membranes, for example, Toyo Membrane Filter (manufactured by Toyo Roshi Kaisha, Ltd.) or Millipore Filter (manufactured by Japan Millipore Limited), or a porous substrate such as activated carbon, etc. Can be appropriately selected and used.

For measurement, if a membrane filter is used for the support, the cell suspension is filtered with a membrane filter,
The cells are held on the filter, the membrane filter is brought into contact with the working electrode 1, and the electrodes are periodically scanned (swept).
This is done by applying a potential and measuring the resulting current. Usually, as the potential scanning, so-called linear scanning in which the potential is changed in proportion to time is preferably adopted.

The current-potential curve thus obtained is the maximum current value (peak current value) proportional to the cell concentration, as will be described later in detail.
In addition to the above, specific information such as the peak potential value and its curve shape which differ depending on the type of cell gives sufficient information for identifying microorganisms such as bacteria.

Therefore, when discriminating the cell type by the method of the present invention, a potential value (peak potential value) that gives a peak current value under specific conditions is obtained for a support carrying a series of standard cells. Then, the peak potential value of the test sample is determined under the same specific conditions as described above, and the peak potential value is compared with that of the standard cell having the same peak potential value. Gram-negative bacteria) can be determined.

Further, when measuring the number of cells by the method of the present invention,
First, a peak current value is obtained under a specific condition for a support carrying a series of known numbers of standard cells, and a calibration curve is prepared for each standard cell. Then, the peak current value of the test sample can be determined under the same specific conditions as above, and the cell number of the test sample can be determined from the calibration curve.

That is, in contrast to the prior art, according to the method of the present invention, the response time and thus the measurement time can be significantly shortened when identifying cells. Furthermore, since the measurement depends on the scanning potential, various disturbing factors in the electrode reaction can be eliminated, which allows more precise and accurate measurement, and at the same time, not only the cell concentration (number) but also its type can be identified. This is a great advantage in practical use such as that which can be done.

Therefore, the present invention can be said to be extremely useful as a sensor means in a wide variety of fields such as a sensor for real-time control of fermentation process, measurement of water quality microbial contamination, measurement of red blood cell and white blood cell count, and the like.

On the other hand, FIG. 2 is a schematic explanatory view showing one example of the apparatus for differential pulse voltammetry which is particularly useful for cell discrimination.

That is, the illustrated apparatus is a working electrode 11 and a counter electrode 12 made of graphite such as plane pyrolytic graphite (hereinafter referred to as BPG), high purity spectroscopic carbon (hereinafter referred to as HPG), carbon, platinum, gold, silver, mercury and the like. And a cell 14 having a reference electrode 13 such as SSCE or SCE, a pulse sequencer 1
5, potential programmer 16, potentiostat 17, drop knocker 18, current-voltage converter 1
9, sample / hold (τ) 110 and same (τ ′) 11
1, a differential amplifier 112 and a recorder 113, and a known system applies and records a differential pulse potential.

For measurement, if a membrane filter is used for the support, the cell suspension is filtered with a membrane filter,
The cells are held on the filter, the membrane filter is brought into contact with the working electrode 11 in the cell 14, and an incremental scanning (sweep) potential with a superposed small potential is applied between the electrodes to measure the differential current generated. Normally, so-called linear scanning, which changes the potential in proportion to time, is preferably used as the potential scanning.

As the minute potential superimposed on the scanning potential, as described above,
Those having an appropriate waveform and period that can give a differential current value can be appropriately selected and used.

The differential current-potential curve obtained in this way gives clear peak potential values that differ depending on the cell type, which not only enables them to be distinguished, but also analyzes the peak waveform of microorganisms such as bacteria. It will also give information on the electrochemical activity of.

That is, according to the method of the present invention, in contrast to the prior art, identification and identification of various bacteria can be easily achieved in an extremely short time.

Here, in the method of the present invention, 4,4′-bipyridine (hereinafter referred to as B, which is an important activator of electron transfer between cells and electrodes).
P) may be used, and the usage conditions thereof are summarized below.

That is, in the present invention, BP is involved in the cell-electrode reaction by being directly added to the buffer solution or fixed on a nitrocellulose membrane or the like and brought into contact with the electrode.

The concentration of BP in the buffer solution varies depending on the type of target cell, but is generally several mM to 100.
About mM is preferable.

As will be shown in Examples below, in the method of the present invention, a scanning potential is applied in the coexistence of BP, and a current value or a differential current value generated by the application of the scanning potential is measured to obtain a peak current value of various cells. Of about 1.5 to 2.5 times, sharpening of the waveform, and sharpening of the peak of the differential current value.

The activator can be omitted when high-precision measurement is not required.

Mechanism of current generation It is considered that electron transfer between the cell and the electrode is closely related to the presence of coenzyme A. Therefore, it is estimated that the peak current value is related to the metabolic pathway.

Therefore, a suspension of Saccharomyces cerevisiae (2.4 x 10
⁸ / ml) to rotenone is a metabolic inhibitor (7.6 m
M), antimycin (5.7 mM), cyanide (1
0.8 mM) and arsenite (10.0 mM) were added, and a scanning potential was applied to measure the current value generated.

As a result, the peak current value was not decreased by the addition of rotenone, antimycin and cyanide salt. On the other hand, a decrease of 4.8 μA to 3.7 μA was observed with the addition of arsenite.

Rotenone specifically inhibits electron transfer in NADH dehydrogenase, antimycin inhibits electron transfer between cytochrome b and cytochrome c, and cyanate inhibits electron transfer between cytochrome oxidase and O _2. Inhibit. On the other hand, arsenite is known to inhibit pyruvate dehydrogenase.

Therefore, it was estimated that the peak current generation was related to the pyruvate dehydrogenase and the citrate cycle.

Further, as shown in the test example described later, the peak current value of the ultrasonically treated cells or cell wall and its eluate obtained by ultrasonically treating the compound existing in the cell wall was measured.

As a result, the peak current value (peak potential value: 0.74 V vs. SSCE) from the cell or cell wall decreased, but the peak current value (peak potential value: 0.65 V) of the eluate.
vs. SSCE) gradually increased.

Since the number of cells is not affected by sonication, the above results indicate that the electrochemically active substance in the cell wall was eluted by sonication and detected electrochemically.

The absorption of the eluate at a wavelength of 260 nm is associated with the adenine ring and increases with the peak current value when the cell suspension is sonicated.

NADH, NADPH, which is a coenzyme having an adenine ring,
FMNH ₂ , FADH ₂ , and coenzyme A can be electrochemically oxidized, but the absorption at a wavelength of 340 nm corresponding to NADH and NADPH and the absorption at a wavelength of 445 to 450 nm corresponding to FMNH ₂ , FADH ₂ are , Not obtained from the eluate.

Further, the half-wave potential value of NADH and NADPH (peak potential value of the present invention) at the carbon electrode is 0.35 to 0.
75V vs. It was in the SCE range. FMNH ₂ and FADH ₂ is, -0.4V vs. It has been reported to be electrochemically oxidized in SCE.

In addition, using the BPG electrode, the current of NADH and coenzyme A-
When the potential curve was obtained, the peak current value of NADH was 0.
35V vs. The peak current value of coenzyme A was 0.65 V vs. SSCE. Each was observed in SSCE.

From the above, it was found that the peak potential value of coenzyme A was similar to that from cells subjected to ultrasonic treatment.

Next, the coenzyme A in the eluate was analyzed by Studman et al. [ER Stadtman et al., Journal of Biological Chemistry, J. Biol. Chem., 191 367 (195).
When enzymatically quantified in 1)], 3.6 mM of coenzyme A was detected.

When cells are sonicated, the concentration of coenzyme A increases with an increase in the peak current value of the eluate, so an increase in the peak current value obtained from the eluate results in an increase in the concentration of coenzyme A in the buffer solution. It is presumed to be due to this.

In other words, it is considered that the coenzyme A, which is an electrode active substance in the cell, comes into contact with the electrode and is oxidized to facilitate the flow of a current, and the peak current value of the oxidation wave (anodic wave anodic wave) appears.

On the other hand, since the coenzyme A existing in the cell wall was eluted in the buffer solution, the peak current value obtained from the cell or cell wall was reduced.

The above results indicate that the coenzyme A present in the cell wall is
It shows that it mediates electron transfer between carbon electrodes. The peak potential value obtained from the eluate is similar to the peak potential value of coenzyme A, and is different from that obtained from whole cells.

The peak potential value is determined by pH, and therefore the above phenomenon is presumed to be based on the difference in pH between the buffer solution and the environment surrounding coenzyme A in the cell wall.

Test example 1. Glucose 4 g, polypeptone 1 g, KH ₂ PO _4
0.5g, the medium 100 ml (pH 7.0) containing MgSO ₄ · 7H 2 O 0.2g Saccharomyces cerevisiae (Saccharomyces cerevisiae), 1 at 30 ° C.
After culturing under aerobic conditions for 8 hours, 5 ° C., 8000 × g
The cells were collected by centrifugation at.

The collected bacterial cells were treated with 0.1 M phosphate buffer (pH 7.0)
Washed twice with 0.1 M phosphate buffer (pH 7.
0) Suspended in 10 ml (cell concentration: 9 × 10 ⁸ cells / m
l) Aerated.

Then, the above-mentioned cultured cells were sonicated for 30 minutes, and then at 5 ° C. for 8
The eluate was obtained by centrifugation at 000 × g and the sonicated cell walls were collected and resuspended in buffer. This cell wall and its eluate were used as samples.

Electrolytic cell (capacity about 25 ml), potentiostat (Model HA301 manufactured by Hokuto Denko KK), linear scanning power supply (Model HB10 manufactured by Hokuto Denko KK)
4) and an XY recorder (RIKEN Denshi F35) were configured as shown in FIG. 1, and this was used as a device for cyclic voltammetry. The working electrode constituting the electrolytic cell was a BPG electrode, a platinum wire electrode was used as the counter electrode, and SSCE was used as the reference electrode.

Each sample was injected into the electrolytic cell of the apparatus for cyclic voltammetry, a cyclic voltammogram was obtained under the condition of 25 ± 2 ° C., and the peak current value of each sample was measured with time.

The results are shown in FIG. 4 [vertical axis: peak current value (μA); horizontal axis: time (minutes)].

In the figure, curve a is the value of the eluate and curve b is the value of the cell wall.

As is apparent from the figure, it is recognized that the electrochemically active substance exists in the cells and can be easily eluted by ultrasonic treatment. The peak potential is not destroyed (Whol
e Cell) 0.74 V vs. SSCE, with an eluent of 0.65 V vs. It is SSCE.

2. Next, when the active substance in the eluate was assayed by the method of Studman or the like, it was confirmed that it was coenzyme A or a similar substance. Therefore, 3.7 mM of coenzyme A prepared separately was used. Similarly, when cyclic voltammetry was performed, the peak potential was 0.65 V vs. the same value as that of the eluate. The value of SSCE was obtained.

From these, it can be seen that the coenzyme A in the bacterial cells affects the peak current.

〔Example〕

Hereinafter, the present invention will be described in more detail by way of examples, but these do not limit the present invention.

Example 1 FIG. 3 shows an example of an electrode system used for cell identification of the present invention.

This electrode system is a membrane filter (10) which is a support for supporting cells (107) to be measured.
8) with a surface area of 0.17 cm ² for producing BPG (105), counter electrode (104) made of platinum wire, SC
E reference electrode (106), potentiostat (Model HA301 manufactured by Hokuto Denko KK) (102), linear scanning power supply (Model HB1 manufactured by Hokuto Denko KK)
04) (101), and XY recorder (RIKEN Denshi F3
5) (103). The electrodes were polished with a polishing cloth soaked with an aqueous suspension of alumina, unless otherwise specified.

A glass electrolytic cell having the above-mentioned three electrodes and separated by a diaphragm (110) is used, and a reference electrode (106) has a porous sintered glass frit (106) whose tip can pass an electrolytic solution.
Separated from the body of the cell by immersion in a glass tube that was joined to the body in a).

Escherichia coli on an agar medium at 37 ° C for 12
After culturing for an hour, 0.1M phosphate buffer (pH 7.0)
Suspended in. Membrane filter (Toyo Membrane Filter TM-2 type, nitrocellulose, pore size 0.
(45 μm, diameter 25 mm), 5 ml of the above-mentioned suspension of cultured cells was dropped with slight suction.

Next, the cell attachment side of this membrane filter was brought into contact with the carbon electrode surface so that the cultured cells came into contact with the carbon electrode. Insert the electrode system in phosphate buffer solution at 25 ± 2 ℃
I got a cyclic voltammogram.

FIG. 5 shows 0 to 1.0 V vs. The cyclic voltammogram at SCE potential is shown [vertical axis: current value (μ
A); horizontal axis: potential (V vs. SCE)].

0.72 V vs. the first scan in the positive direction.
The peak current value of the anode wave appeared in SCE. No corresponding decrease peak was seen during the reverse scan.

Therefore, the cell electrode reaction is irreversible.

In the case of this example, the peak current value in the range of 0.1 to 1.9 × 10 ⁹ cells / ml was proportional to the number of cells on the membrane filter.

This result shows that Escherichia coli (Escheric
It means that the number of cells (hia coli) can be obtained from the peak current value of cyclic voltammetry. The minimum detectable number of Escherichia coli cells is 5 x
It is 10 ⁷ cells / ml (suspension equivalent).

Example 2 Using the same electrode system as in Example 1, Bacillus subtilis (Gram positive), Lactobachillus fermentum (Lactobachillus fermentum;
Gram positive), Streptococcus sanguis (Strept
ococcussanguis; Gram positive), Staphylococcus epidermidis (Gram positive), Escherichia coli (Gram negative), Salmonella typhimurium (Salmonella typ)
hymurium; Gram-negative) 6 × 10 ^{8 of} each were held on the same membrane filter as in the previous example, and the potential at which the peak current value appeared by each cyclic voltammetry was measured. The results are as shown in Table 1 below.

Bacillus subtilis, Lactobachillus fermentu
m), Streptococcus sangis (Streptococcus s)
anguis), Staphylococcus epidermidis (S
The cell membrane of Gram-positive bacteria such as taphylococcus epidermidis) is surrounded by a cell wall (typically about 250 Å in thickness) composed of peptidoglycan and decoylic acid. The structure of the cell wall and the cell membrane of Gram-negative bacteria such as Escherichia coli and Salmonella typhymurium is more complicated, and the cell membrane is surrounded by the wall of peptidoglycan having a thickness of about 30 Å, and the wall is further outside. Is covered with a mosaic membrane of proteins, lipids and lipopolysaccharides about 80 Å thick.

The above results indicate that the cell wall structure influences the peak potential value of cyclic voltammetry.

That is, the peak current of Gram-negative bacteria covered with a more complicated membrane appeared at a potential on the positive side of that of Gram-positive bacteria.

Example 3 Bacillus s that is a Gram-positive bacterium
ubtilis) IFO3009 in nutrient medium (Nutrient Brot
h, beef extract 1%, peptone 1%), harvested in the logarithmic phase, and 0.1M phosphate buffer (pH 7.0)
After washing with, the cell suspension (1.2 x 10
⁹ / ml) was prepared.

Similarly, gram-positive bacteria Saccharomyces cerevisiae (YRD medium: yeast extract 1%, polypeptone 2%, glucose 2%), gram-positive bacteria Lactobacillus fermentum (La
ctobachillus fermentum) IFO3071 (nutrient medium), gram-positive bacterium Leuconostoc mesenteroides IFO383
2 (tomato juice medium: 1% tryptone, 1% yeast extract, 20% tomato juice) and Gram-negative bacteria Escherichia coli (nutrient medium) were respectively cultivated and collected in the logarithmic phase and added to the buffer solution. Suspension was performed to prepare suspensions having viable cell count concentrations of 1.03 × 10 ⁹ , 5.0 × 10 ⁸ , 1.3 × 10 ⁹ and 1.0 × 10 ¹⁰ cells / ml.

Next, each of these cell suspensions was dropped onto the same membrane filter as in the previous example while slightly sucking each 5 ml, and the working electrode B having the membrane filter attached to the surface thereof.
Using PG, counter electrode platinum wire and reference electrode SSCE,
Scanning potential 0 to 1.0 V vs. SSCE, sampling time 20 ms, modulation voltage 50 mV and 100 m
Differential pulse voltammetry was performed under the conditions of V, potential single sweep 0.5 mV / sec, and measurement temperature 25 ° C. The device used was "Polarograph Model 312" manufactured by Fuso Seisakusho.

Figure 6 shows Bacillus subtilis
Is a differential pulse voltammogram of 0.68 V vs.
An extremely clear peak potential is observed in SSCE (in the figure, symbols (a) and (b) correspond to modulation voltages 100 mV and 50 mV, respectively).

The peak potential values of each bacterium are summarized in Table 2 below.

As is clear from the same table, according to the method of the present invention, the distinction between bacteria can be made extremely clearly and easily by differential pulse voltammetry, and particularly Escherichia coli which is a gram-negative bacterium and its It is worth noting that the other Gram-positive bacteria can be clearly distinguished.

Example 4 1. Microorganism Saccharomyces cerevisi
ae) is glucose 4 g, polypeptone 1 g, KH ₂ P
O ₄ 0.5 _g, aerobically 30 ° C. to培値(pH 7.0) in 100ml containing MgSO ₄ · 7H 2 O 0.2g 1
It was cultured for 2 hours.

Salmonella typhymuri
um) TA100 was cultivated aerobically at 30 ° C. for 15 hours in 100 ml of a medium containing 1 g of polypeptone and meat extract.

Escherichia coli K12 contains glucose 0.
1 g, Bacto tryptone 1 g, yeast extract 0.5 g,
And 100 ml of medium containing 5 g of NaCl (pH 7.
0) was aerobically cultured at 30 ° C for 12 hours.

Lactobachillus f.
ermentum) ATCC 9338 is a tripty case 1g,
Tryptose 0.3 g, yeast extract 0.5 g, KH ₂ P
O ₄ 0.3 g, K ₂ HPO ₄ 0.3 g, ammonium citrate 0.2 g, glucose 2 g, Tween 80
(Registered trademark) 0.1 g, cysteine hydrochloride 0.02 g,
And 0.5ml of the solution _{(MgSO 4 · 7H 2 O 11} .
_{5%, FeSO 4 · 7H 2} O 0.68%, MnSO 4
-The cells were anaerobically cultivated at 37 ° C for 16 hours in 100 ml of a medium (pH 6.8) containing 2H ₂ O containing 2.4%.

Bacillus subtilis MI11
2 is K ₂ HPO ₄ 1.4 g, KH ₂ PO ₄ 0.6
g, (NH ₄ ) ₂ SO ₄ 0.2 g, sodium citrate 0.1 g, casamino acid 0.5 g, glucose 0.5
10 containing g, and MgSO ₄ · _7H 2 O 0.02g
It was aerobically cultured in 0 ml of medium (pH 7.0) at 30 ° C. for 15 hours. 2.4,4'-bipyridine modified electrode 1.54 g of 4,4'-bipyridine was dissolved in 100 ml of methanol to adjust the concentration of the solution to 100 mM. 0.3
A BPG electrode (0.17c) that has been previously polished with a polishing cloth soaked with an aqueous alumina suspension having a particle size of μm.
m ² ) was immersed in the above solution and rocked gently for 1 minute.

As described above, the BPG electrode was polished before each operation, and each time, it was also modified with 4,4'-bipyridine.

3. Measurement method Potentiostat (Model manufactured by Hokuto Denko Co., Ltd.
HA301), linear scanning power supply (Hokuto Denko Co., Ltd.
Cyclic voltammetry was carried out using a Model HB104) and an XY recorder (Riken Denshi F35) and a 4,4′-bipyridine modified carbon electrode.

Cyclic voltammetry electrolysis cell has a volume of 25 ml
The cell is made of glass, and the counter electrode is a platinum wire and the reference electrode is SCE. The reference electrode is isolated from the body of the electrolysis cell by immersion in a glass tube whose tip is joined to the body with a sintered glass frit.

Membrane filter (Toyo Membrane Filter TM-
2 types, nitrocellulose, pore size 0.45μm, diameter 2
5 mm) and various cells to be measured were 1.1 × 10 ⁹
Individually held, this membrane filter was attached to the 4,4'-bipyridine modified carbon electrode, and the bacterial cells were brought into contact with the electrode.

In addition, the measurements were made by Fuso Seisakusho “Polarograph 312
Differential pulse voltammetry was performed under the same conditions as the cyclic voltammogram using a “type” and XY recorder to obtain a differential pulse voltammogram.

FIG. 7 shows a cyclic voltammogram and a differential pulse voltammogram for Saccharomyces cerevisiae at pH 7.0.
In the figure, (A) shows a cyclic voltammogram, (B) shows a differential pulse voltammogram, (a) relates to a 4,4'-bipyridine modified electrode, and (b) is an electrode not modified with 4,4'-bipyridine. It is about. The magnitude of the current value is indicated by the width of the arrow and the current value corresponding to the width.

The measurement was performed at 25 ± 2 ° C. The cyclic voltammogram has a scanning speed of 10 mV / sec, and the differential pulse voltammogram has a scanning speed of 10 mV / sec and a wave height of 100 mV.
And a pulse width of 20 msec.

Upon the first scanning in the positive direction, both the modified electrode and the unmodified electrode were 0.74 V vs. Anodic waves were observed in SCE. The peak potential value of the cyclic voltammogram and that of the differential pulse voltammogram match.

The peak current value obtained from the 4,4'-bipyridine modified electrode was higher than the peak current value obtained from the unmodified electrode. No peak current was generated in the absence of cells.

Therefore, it can be seen that the electrode is activated by the 4,4′-bipyridine modification.

When the peak potentials of the various microorganism samples were measured, Escherichia coli 0.72V vs S
CE, Salmonella typ
hymurium) 0.70 V vs. SCE, Bacillus subtilis 0.68V vs. SC
E, Lactobachilles fermentum
us fermentum) 0.68V vs. It was SCE.

Example 5 Bacillus subtilis MI11
2 (Arg-15, leu B8 thi5 r - m -
rec E4, hereinafter referred to as plasmid-free in this Example) is 100 m of Spitzen medium.
l, and also Bacillus subtilis
s) MI112 (containing plasmid PTL12; hereinafter referred to as plasmid in this Example) is a spitzen medium containing 1 μg / ml of trimethoprim.
(Spizizen Medium) Inoculate 100 ml of each to 37
The cells were cultured aerobically at 12 ° C for 12 hours. After collecting the cells, the cells were washed with 0.1 M phosphate buffer (pH 7.0) and suspended to make the cells B
The surface of the PG electrode was brought into contact with the membrane filter.

The measurement was performed by using a 20 ml H-type cell as an electrolysis cell, BPG (0.17 cm ² ) as a working electrode, a platinum wire as a counter electrode, and SCE as a reference electrode to measure electrochemical behavior by cyclic voltammetry. , And asked for the cyclic voltammogram. The potential scanning speed was set to 10 mV / sec, and the measurement was performed at 25 ° C.

As a result of obtaining the cyclic voltammograms with and without the plasmid, the peak potential at which the peak current was generated in each cell was obtained as shown in Table 3 below.

This indicates that cells containing the plasmid can be measured in the same manner.

Example 6 Lactobachillus fermentum
mentum), Staphylococcus epidermidis (St
aphylococcus epidermidis), Streptococcus sanguis, Escheric
hia coli), Salmonella typhimurium (Salmonel
la typhymurium), Proteus bulgaris
vulgaris) in Rogosa medium at 37 ℃ for 16 hours,
A stationary culture was performed aerobically.

An appropriate amount of each colony was scraped off, suspended in 0.1 M phosphate buffer (pH 7.0), and this was dropped on a membrane filter to collect bacterial cells on the filter, and the BPG electrode surface (0. 17 cm ² ) to make a working electrode. Platinum wire was used as the counter electrode, SCE was used as the reference electrode, H-type cell containing 0.1 ml of 0.1M phosphate buffer solution (pH 7.0) was used as the electrolytic cell, and electrochemical behavior was determined by cyclic voltammetry. It measured and calculated | required the cyclic voltammogram.

The measurement results of the peak potential for each sample are shown in Table 4 below.

From this result, it can be seen that even if the cell numbers are not the same, a specific peak potential is exhibited within a certain range.

Example 7 Bacillus subtilis IFO3
009, Saccharomyces
cerevisiae), Lactobacillus fermentum (Lact)
obachillus fermentum) IFO3071, Leuconostoc mesenteroides
) IFO3832 and Escherichia col
i) was cultivated under the same conditions as in Example 2 above, and the cells were collected in the logarithmic phase to prepare samples of 1.3 × 10 ⁹ viable cells / filter.

Next, for each of these samples held on the filter, differential pulse voltammetry was carried out under the same conditions as in Example 3 using the 4,4′-bipyridine modified BPG electrode used in Example 4 above. .

The peak potential values of each sample are summarized in Table 5.

As is clear from the table, according to the method of the present invention, it is possible to distinguish cells from each other very clearly and easily.

Example 8 Saccharomyces cerevisiae cultivated aerobically for 12 hours was collected, and then suspended in a 0.1 M phosphate buffer solution (pH 7.0) to form a monolayer of the cells. After immobilization on the membrane filter, the bacterial cells were brought into contact with the surface of the BPG electrode.

0.1M phosphate buffer solution (pH) in the electrolysis cell (25 ml H type)
7.0), BPG was used as a working electrode, a platinum wire was used as a counter electrode, and SCE was used as a reference electrode, and potential scanning was performed by cyclic voltammetry. As a result, 0.74 V v
s. A specific current peak was obtained near SCE.

[Brief description of drawings]

FIG. 1 is a schematic explanatory view of an apparatus for cyclic voltammetry used in the present invention, and FIG. 2 is a schematic explanatory view of an apparatus for differential pulse voltammetry used in the present invention,
FIG. 3 is a schematic explanatory view of an example of an electrode system used for cell identification, FIG. 4 is a graph showing changes in peak potential values with time, and FIG. 5 is a cyclic voltammogram of Escherichia coli. FIG. 6 is a differential pulse voltammogram of Bacillus subtilis, and FIG. 7 is a cyclic voltammogram (A) and differential pulse voltammogram (B) of Saccharomyces cerevisiae. 4 ... Electrolysis cell, 5 ... Potentiostat, 6 ... Linear / sweave power supply, 7 ... XY recorder or Shincross Cove, 11 ... Working electrode, 12 ... Counter electrode, 14 ... Electrolysis cell, 15 ... Pulse sequencer, 17 ... Potentiostat, 112 ... Differentiation amplifier and 113 ... Recorder

Claims (3)

[Claims] 1. In a solution in which a working electrode and a counter electrode are immersed, cells or cell wall pieces accumulated on a support are brought into contact with the working electrode, a scanning potential is applied to the working electrode, and a scanning potential is applied. The method for electrochemically identifying a cell, which comprises the step of measuring a maximum value of a current value or a differential current value generated by the above or a scanning potential that maximizes the current value or the differential current value. 2. A cell or cell wall piece accumulated on a support is brought into contact with the working electrode in a solution in which the working electrode and the counter electrode are immersed, and a scanning potential is applied to the working electrode, and a scanning potential is applied. The method for electrochemically discriminating cells, comprising a step of measuring a scanning potential that maximizes a current value or a differential current value generated by, and comparing the scanning potential with a corresponding scanning potential of a standard cell measured in advance. . 3. A solution in which a working electrode and a counter electrode are immersed, and cells or cell wall pieces accumulated on a support are brought into contact with the working electrode, and a scanning potential is applied to the working electrode, and a scanning potential is applied. It is characterized by including the step of measuring the maximum value of the current value or the differential current value generated by, and comparing with the correlation between the maximum value of the current value or the differential current value and the number of cells measured in advance. Electrochemical counting method of cells.